Ferroptosis is a novel form of programmed cell death characterized by the accumulation of lipid peroxides and associated with neuropathic diseases. However, the molecular mechanisms remain unclear. This study aimed to investigate the potential mechanism of DDX3 in pentylenetetrazole (PTZ)-induced ferroptosis in human neuronal SH-SY5Y cells. PTZ induced SH-SY5Y cells to simulate the neuropathic disease model in vitro. Western blot analysis was used to assess DDX3, β-catenin, β-catenin phosphorylated at Ser37/Thr41, GPX4, and ACSL4 expression. Nuclear accumulation of β-catenin was tested by IF. MMP-7, c-Myc, cyclin D1, LEF1, and Axin2 were detected by qRT-PCR. Cell viability was measured by CCK-8. Apoptosis was detected by flow cytometry. Total antioxidant status (TAS) and total oxidant status (TOS) levels were detected by biochemical kit. ROS production was detected by flow cytometry. Biochemical kits were used to detect MDA, 4-HNE, Fe2+, and GSH levels. Our results showed that DDX3 expression was decreased in PTZ-induced SH-SY5Y cells. DDX3 overexpression promoted PTZ-induced SH-SY5Y cell viability, inhibited apoptosis, promoted TAS and GSH expression, and inhibited TOS, MDA, 4-HNE, Fe2+, and ROS levels, indicating that DDX3 reduced PTZ-induced SH-SY5Y cell ferroptosis. DDX3 knockdown reduced total β-catenin protein, nuclear accumulation of β-catenin, Wnt target genes (MMP-7, c-Myc, cyclin D1, LEF1, and Axin2), and GPX4 expression in PTZ-induced SH-SY5Y cells, while increasing β-catenin phosphorylated at Ser37/Thr41 and ACSL4 expression. The effect of DDX3 overexpression on the above indexes was opposite to that of DDX3 knockdown. β-catenin overexpression and Wnt/β-catenin signaling activator CHIR99021 increased total β-catenin protein, nuclear accumulation of β-catenin, MMP-7, c-Myc, cyclin D1, LEF1, and Axin2 expression in PTZ-induced SH-SY5Y cells, while decreasing β-catenin phosphorylated at Ser37/Thr41expression and ACSL4 expression. In addition, β-catenin overexpression and CHIR99021 increased cell viability, reduced apoptosis, and upregulated TAS, GSH, and GPX4 expression, while decreasing TOS, MDA, 4-HNE, Fe2+, ROS, and ACSL4 levels. GPX4 knockdown and ACSL4 overexpression reversed β-catenin overexpression effects. Further results showed that DDX3 inhibited PTZ-induced SH-SY5Y cell ferroptosis by activating Wnt/β-catenin signaling. Our results suggested that DDX3 inhibited PTZ-induced ferroptosis in SH-SY5Y cells through activation of Wnt/β-catenin signaling. Our findings may provide new molecular targets for the treatment of neuropathic diseases.

Background This study aimed to develop and validate a practical and reliable tool for comprehensive assessment of brain lesions on immediate post‐endovascular therapy (EVT) noncontrast computed tomography (CT) and to predict 90‐day functional outcomes in patients with acute ischemic stroke. Methods We retrospectively reviewed consecutive patients with acute ischemic stroke undergoing EVT from 2 academic hospitals as the development cohort and used an independent national multicenter prospective cohort for external validation. The new score was derived from the Alberta Stroke Program Early CT Score (ASPECTS) on the immediate post‐EVT non‐contrast CT, integrating both hyperdensity and hypodensity signs, named comprehensive Alberta Stroke Program Early CT Score (Co‐ASPECTS). Unfavorable outcome was defined as modified Rankin Scale score of 3 to 6 at 90 days. Results A total of 1015 patients were included (development cohort: 542; validation cohort: 473). Co‐ASPECTS, formulated through equal‐weight summation of hyperdensity and hypodensity signs and consistent with the 0 to 10 score ASPECTS framework, was significantly associated with unfavorable outcomes (adjusted odds ratio, 0.45 [95% CI, 0.40–0.51]). It showed discrimination for functional outcome with a C‐statistic of 0.889 in the development cohort, outperforming conventional models including baseline ASPECTS and immediate post‐EVT ASPECTS that calculated based on hyperdensity or hypodensity signs alone. In the validation cohort, Co‐ASPECTS maintained robust performance (C‐statistic, 0.847). It also appeared to reflect both infarct burden and secondary injury, showing associations with final infarction and intracranial hemorrhage. Conclusions Co‐ASPECTS may serve as a promising tool with reliability and practicality for assessing brain lesions and predicting functional outcomes of patients with acute ischemic stroke who underwent EVT in clinical practice.

Background: Early neurological deterioration (END) is a common complication following intravenous thrombolysis in acute ischemic stroke (AIS). This study aimed to identify factors associated with END to improve early risk stratification and management. Methods: We conducted a prospective observational study including AIS patients treated with intravenous recombinant tissue plasminogen activator (rt-PA) within 4.5 hours of symptom onset. END was defined as an increase of >= 4 points in the NIHSS score within 24 hours after thrombolysis. Baseline data included demographics, vascular risk factors, prior medication use, NIHSS score, onset-to-needle and onset-to-thrombectomy times, laboratory markers (including inflammatory cytokines and platelet function), and neuroimaging findings. Univariate and multivariate logistic regression analyses were performed, and model performance was evaluated using receiver operating characteristic (ROC) curves. Results: Among 300 enrolled patients, 66 (22.0%) developed END. Multivariate analysis identified higher NIHSS score at admission (P=0.011), longer onset-to-needle time (P=0.007), proximal large vessel occlusion (P=0.010), diabetes (P=0.018), elevated interleukin-6 (IL-6) levels (P<0.001), and increased maximum aggregation rate induced by arachidonic acid (MAR_AA) (P<0.001) as independent predictors of END. A predictive model incorporating these factors demonstrated excellent discriminative ability (AUC=0.873; 95% CI: 0.844-0.902). Conclusion: Neurological severity, treatment delay, metabolic vulnerability, inflammation, and platelet hyperreactivity collectively contribute to END after thrombolysis. Identifying high-risk patients through clinical and biomarker profiling may help improve outcomes through early intervention.

BACKGROUND:Ischemic stroke (IS) is an acute cerebrovascular disease characterized by high morbidity and mortality, with limited current treatment options. Tumor protein p53-inducible nuclear protein 2 (Tp53inp2) is known to be a positive regulator of autophagy under physiological conditions, but the mechanism of Tp53inp2 in IS remains unclear. In this study, we aimed to explore the mechanism of Tp53inp2 in IS. METHODS:Primary neural stem cells (NSCs) were extracted and identified. An OGD/R cell model was constructed. Tp53inp2 was knocked down and rapamycin was added. A middle cerebral artery occlusion (MCAO) animal model was constructed, and then 5 μL of 5 × 105 NSCs, either untreated or transfected with sh-NC or sh-Tp53inp2, were injected. Additionally, at the cellular level, Ptgs2 or Tp53inp2 was overexpressed, and METTL14 was knocked down. RESULTS:Inhibition of Tp53inp2 mitigated OGD/R-induced mitophagy and ROS levels in vitro. Moreover, inhibition of Tp53inp2 mediated neuronal differentiation of OGD/R treated NSCs by suppressing mitophagy. At the animal level, the transplantation of NSCs with a knockdown of Tp53inp2 increased neuronal differentiation, thereby alleviating the effects of MCAO and mitigating cognitive impairments in MCAO model mice. Ptgs2 was further screened and validated as a downstream target mediating the effect of Tp53inp2 on NSCs. At the cellular level, Tp53inp2 alleviated OGD/R-induced mitophagy and ROS levels by regulating Ptgs2 expression. METTL14 could regulate Tp53inp2 expression by modulating the functional m6A modification sites on Tp53inp2 mRNA. Inhibition of METTL14 alleviated OGD/R-induced mitophagy and the rise of ROS levels in NSCs by inhibiting the Tp53inp2/Ptgs2 axis. CONCLUSIONS:Inhibition of METTL14 alleviated OGD/R-induced neuronal differentiation injury in NSCs by inhibiting the Tp53inp2/Ptgs2 axis. By elucidating the mechanisms involving Tp53inp2, Ptgs2, and METTL14, this research offers a foundation for developing new strategies to enhance neuronal differentiation and mitigate cognitive impairments in stroke patients.

Background Parkinson's disease (PD), a common neurodegenerative condition, afflicts patients through the progressive degeneration of dopaminergic neurons and sustained neuroinflammation. This study investigates the role of olfactory mucosa-derived mesenchymal stem cell (OM-MSC)-derived exosomes, particularly the long non-coding RNA A2M-AS1 (lncA2M-AS1), in modulating microglial metabolism reprogramming and neuroinflammation in PD. Methods A mouse PD model was established using MPTP injections. Animals received treatments including OM-MSC-derived exosomes knockdown for lncA2M-AS1 or AAV-mediated lncA2M-AS1 overexpression. Motor function was assessed using the open field test and the apomorphine-induced rotation test. Glycolytic metabolism was evaluated by measuring ECAR and OCR using Seahorse XFp Analyzer, and the expression of glycolytic proteins (GLUT1, HK2, PKM2, LDHA) via Western blot. Molecular analyses included qPCR, Western blot, Co-IP, and ubiquitination assays that were performed to investigate the lncA2M-AS1/CFL1/ROCK1 regulatory axis. Histological examinations involved immunohistochemistry for TH and IBA1. The expressions of lncA2M-AS1 and ROCK1 were determined in serum obtained from individuals with PD and matched controls. Results LncA2M-AS1 is downregulated in PD patient serum and MPTP mice. OM-MSC exosomal lncA2M-AS1 suppressed microglial glycolysis, reduced pro-inflammatory cytokine release, enhanced neuronal viability, and improved motor function in PD mice. Mechanistically, lncA2M-AS1 directly binds to CFL1 mRNA, promoting ubiquitin-mediated degradation of ROCK1 and inhibiting the CFL1/ROCK1 pathway. Knockdown of CFL1 or overexpression of lncA2M-AS1 attenuated microglial activation and neuroinflammation, whereas ROCK1 overexpression reversed these protective effects. Conclusion OM-MSC exosomal lncA2M-AS1 ameliorates PD pathogenesis by targeting the CFL1/ROCK1 axis to reprogram microglial glucose metabolism and suppress neuroinflammation, offering a novel therapeutic strategy for PD.

Sleep deprivation (SD) has become a critical global health concern, with consequences including cognitive decline. Transcranial alternating current stimulation (tACS) holds significant potential for improving cognitive impairment. This study aimed to evaluate the effects of tACS on SD-associated cognitive deficits. Male C57BL/6 mice were subjected to SD using the horizontal platform method. Cognitive function was assessed via multiple behavioral tests, and local field potential (LFP) signals were recorded. Untargeted metabolomics was employed to identify key metabolites and regulatory pathways in mouse brain tissue. Mouse models and co-culture systems were combined to investigate the regulatory relationships among microglia, synapses, and neurons. tACS significantly improved memory and cognitive function in SD-induced model mice, accompanied by inhibited microglial phagocytosis, increased synaptic density, and reduced neuronal apoptosis. Untargeted metabolomics revealed that tACS may regulate nervonic acid (NA) abundance and the Rap1 signaling pathway during SD. Further studies demonstrated that NA suppresses lipopolysaccharide (LPS)-induced microglial overactivation and synaptic loss via the Rap1 signaling pathway, thereby blocking neuronal apoptosis. Additionally, similar to the therapeutic effects of tACS, NA administration markedly alleviated cognitive impairment and neural damage in SD mice. These data support the regulatory roles of tACS and NA in neuronal activity and synaptic plasticity and propose novel therapeutic targets for SD-related cognitive deficits.

Exosome Lnc A2M-AS1 from olfactory mucosa mesenchymal stem cells (OM-MSCs) can ameliorate oxidative stress by improving mitophagy in cardiomuscular cells; however, it remains unclear whether this effect exists in the brain tissues of patients with Parkinson’s disease (PD). OM-MSC–Exosomes were isolated and verified based on morphology and specific biomarkers. The effects of OM-MSC-Exo on mitochondrial autophagy, oxidative stress, and lncRNA A2M-AS1 were detected in MPP+-treated HT22 cells. The effects of OM-MSC-Exos on mitochondrial autophagy and oxidative stress were detected in an MPTP-induced Parkinson's disease (PD) model in C57BL/6 mice. The interaction between IGF2BP1, A2M-AS1, and TP53INP1 was assessed via RNA pull-down/RNA Immunoprecipitation and RNA stability assays. The effects of lnc A2M-AS1 on IGF2BP1/TP53INP1-mediated mitochondrial autophagy and oxidative stress were verified in MPP+-treated HT22 cells and MPTP-induced PD mouse models. Exosomes isolated from olfactory mucosa mesenchymal stem cells were found to be rich in Lnc A2M-AS1. Lnc A2M-AS1 was proved to be able to ameliorate oxidative stress induced by MPP+ in HT22 cells. lncRNA A2M-AS1 regulates oxidative stress by enhancing mitophagy in HT22 cells. In addition, lncRNA A2M-AS1 induced mitophagy through TP53INP1 and mediated TP53INP1 expression by binding to IGF2BP1. Furthermore, OM-MSC-Exo and Lnc A2M-AS1 treatment improved symptoms and ameliorated oxidative stress in MPTP-induced PD mouse models. Collectively, lncRNA A2M-AS1 from OM-MSC-derived exosomes regulates TP53INP1 expression by targeting IGF2BP1 to induce mitophagy and ameliorate oxidative stress. OM-MSC-derived exosomes could potentially serve as promising candidates for new treatment methods for PD.

OBJECTIVE:Chronic insomnia can easily lead to clinical distress or cause mental, social, physical, educational, occupational, or other functional impairments. Considering the role of circadian rhythm in insomnia, we focused on exploring the action of miR-29a in regulating the PER2 gene in improving chronic insomnia. METHODS:LPS induces the expression of miRNAs targeting PER2 in HMC3 and PRM cells, which was verified by RT-qPCR. Poly-lactic-co-glycolic acid (PLGA) nanoparticles (NP) were used to encapsulate short hairpin (sh)-miR-29a to construct sh-miR-29a-NP. Morris water environment method was employed to establish a sleep deprivation rat model to investigate the therapeutic effects of sh-miR-29a-NP. Cell viability and levels of cell polarization factors were evaluated using CCK8 and ELISA, respectively. The Morris water maze test was applied to assess the learning and memory capabilities of the rats. Immunohistochemistry, immunofluorescence, and western blot were applied to test the expression of glial cell polarization, neuronal cell activation, apoptosis, and Period2 (PER2) /nuclear factor kappa B (NF-κB) axis proteins. RESULTS:miR-29a was significantly upregulated in LPS-induced HMC3 and PRM cells, with the most significantly altered miRNAs/PER2 interaction. In LPS-induced HMC3 and PRM cells, sh-miR-29a promoted the PER2 and CD206 expressions, and inhibited the expression of ionized calcium-binding adaptor molecule 1 (IBA-1), NF-κB, and CD86, while this effect was blocked by small interfering-PER2. Further in vivo experiments confirmed that PER2 and CD206 expression was reduced, while NF-κB, CD86, and IBA-1 expression were up-regulated in the hippocampal tissue of CSD rats. However, this effect was reversed by treatment with sh-miR-29a-NP. Treatment with sh-miR-29a-NP in CSD rats shortened the escape latency and increased the number of crossings over the original platform, while inhibited the expression of NLR Family Pyrin Domain Containing 3, caspase-1, Gasdermin D (GSDMD), and TUNEL signal in the hippocampal tissue. CONCLUSION:The regulation of PER2/NF-κB pathway by sh-miR-29a-NP promoted M2 polarization of microglial cells and inhibited neuronal cell pyroptosis, thereby improving cognitive dysfunction in chronic insomnia.

Nobiletin (NOB), a naturally occurring polymethoxyflavonoid, has been shown to regulate the expression of the clock gene BMAL1. This study aims to explore the impact of NOB nanoparticles on microglial polarization and cognitive impairments resulting from chronic sleep paradoxical deprivation (PSD), as well as the mechanisms involved. Following PSD modeling, rats treated with NOB nanoparticles exhibited significantly improved cognitive performance in behavioral tests. The treatment upregulated the expression of BMAL1, SIRT1, E2F1, and the NAD+/NADH ratio, shifted microglial polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, and enhanced antioxidant defenses. The NAD+ inhibitor apocynin and silencing of BMAL1 could reverse the effects of NOB nanoparticles. Overexpression of BMAL1 had similar effects to NOB nanoparticles in LPS-induced cellular models, while silencing of SIRT1 or E2F1 reversed the effects. Co-immunoprecipitation experiments illustrated the binding of SIRT1 and BMAL1 and SIRT1 and E2F1. NOB nanoparticles alleviate chronic PSD-induced microglia M1 polarisation, inflammation, and cognitive deficits in rats by a mechanism that may be related to the BMAL1/SIRT1/E2F1 axis, providing a new direction for the therapeutic approach of chronic PSD-associated cognitive deficits.

BACKGROUND:Cerebral ischemia-reperfusion (CI/R) injury, a major complication of ischemic stroke, is characterized by mitochondrial dysfunction and neuronal apoptosis, and understanding its underlying molecular mechanisms is essential for the development of effective therapeutic strategies. This study aimed to investigate the role of ubiquitin-specific protease 7 (USP7) in CI/R injury and elucidate its regulatory mechanisms. METHODS:A rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) and an in vitro neuronal model subjected to oxygen-glucose deprivation/reperfusion (OGD/R) were used to mimic CI/R injury. USP7 was overexpressed or knocked down, with or without co-treatment, using the autophagy inhibitor 3-methyladenine (3-MA). Neurological function was evaluated using standardized scoring systems, and cerebral infarct volume was quantified by TTC staining. Histopathological changes in the cortex and hippocampus were assessed using hematoxylin-eosin (HE) and Nissl staining. Neuronal viability and apoptosis were measured by CCK-8 assay, TUNEL staining, and flow cytometry. To assess cellular metabolism and oxidative stress, ATP and LDH levels, along with antioxidant markers (including SOD, GSH, and GSH-Px), were analyzed using commercial biochemical kits. Mitochondrial morphology and autophagosome formation were visualized using transmission electron microscopy. Gene and protein expression levels were quantified by qRT-PCR and Western blotting, respectively. Immunofluorescence microscopy was performed to evaluate the subcellular localization of target proteins and co-localization with mitochondrial membrane markers. Lastly, protein-protein interactions and ubiquitination modification were analyzed by co-immunoprecipitation assays. RESULTS:USP7 overexpression significantly alleviated neurological deficits, reduced infarct volume, attenuated histological damage, and decreased neuronal apoptosis in the MCAO/R model. Similarly, in the OGD/R model, USP7 overexpression markedly enhanced neuronal viability, suppressed apoptosis, restored ATP production, improved antioxidant capacity (as evidenced by increased levels of SOD, GSH, and GSH-Px), and reduced LDH release. Mechanistically, USP7 stabilized SIRT1 protein expression through deubiquitination, which in turn activated the PINK1/Parkin pathway and enhanced mitophagy. This activation was demonstrated by an increased LC3II/LC3I ratio, elevated ATG5 expression, enhanced co-localization of Tomm20 and Parkin, and increased autophagosome formation. Moreover, these protective effects were abolished when either 3-MA treatment was applied or SIRT1/PINK1 expression was knocked down. CONCLUSION:USP7 mitigates CI/R injury by promoting PINK1/Parkin-dependent mitophagy through SIRT1 deubiquitination and stabilization, suggesting USP7 as a potential therapeutic target for ischemic stroke.

5-Methoxytryptophan (5-MTP), a candidate biomarker for chronic kidney disease (CKD), has an undefined role in cerebrovascular pathophysiology. To investigate this, we employed a folic acid (FA)–induced CKD to simulate cerebrovascular complications in vivo. Additionally, in vitro models of cerebral ischemia and cerebrovascular endothelial cell injury were established. 5-MTP was administered to rats and cells, along with nuclear factor-κB (NF-κB) expression. The pathological characteristics of kidney and brain tissue were observed by histological staining. Cell proliferation was assessed using the Cell Counting Kit 8, while tube formation and migration were examined using tube formation and wound healing assays. Cell apoptosis was detected using both TdT-mediated dUTP-biotin nick end labeling and flow cytometry. Levels of renal injury markers, blood biomarkers of cerebrovascular disease, and inflammatory cytokines were measured using biochemical assays. Quantitative real-time PCR and Western blot were used to detect the mRNA and protein expression, respectively. Key findings revealed that FA successfully induced CKD in rats, which subsequently exacerbated cerebrovascular dysfunction. 5-MTP reduced the levels of proteinuria, N-acetyl-beta-d-glucosaminidase, nephrin, endothelin-1, von Willebrand factor, and thrombomodulin; improved the degree of renal fibrosis and structural damage to the brain tissue; and inhibited cell apoptosis in rats. In vitro, 5-MTP promoted cell proliferation, tube formation, migration, and the upregulation of B-cell lymphoma-2 and caspase-3 expression. This treatment also led to an increase in interleukin (IL)-10 levels while suppressing cell apoptosis, Bcl-2-associated X protein (Bax), and cleaved caspase-3 expression. Furthermore, it reduced the IL-6 and tumor necrosis factor-alpha levels. NF-κB overexpression reversed the effects of 5-MTP in vitro and in vivo. Our results demonstrate that 5-MTP ameliorated CKD-induced cerebrovascular injury through the NF-κB pathway, indicating its potential as an innovative and efficacious therapeutic target for CKD-induced cerebrovascular dysfunction.

BACKGROUND:Circadian rhythm sleep-wake disorders (CRSD) and cognitive impairment are common features of many neurological disorders. Suvorexant is a novel drug for the treatment of insomnia. The study aimed to investigate the effects and potential mechanisms of suvorexant in alleviating CRSD. METHODS:The photothermal displacement-induced CRSD model rats were treated with suvorexant to investigate its impact on cognitive function, the BMAL1/NLRP3 axis-related genes, the microglial marker IBA1, Aβ levels, and the expression of inflammatory factors. Subsequently, hippocampal delivery of sh-BMAL1 was used to investigate the specific mechanism by which suvorexant alleviates the CRSD model. RESULTS:Suvorexant treatment significantly decreased sleep latency and escape latency, while increasing sleep time, swimming speed, the number of crossings, and the time spent in each quadrant in the CRSD group. Moreover, Suvorexant was found to upregulate the levels of BMAL1, IL-10, CD206, and TGF-β in CRSD rats, and downregulate the levels of NLRP3, IL-1β, Aβ1-40, Aβ1-42, IL-6, TNF-α, and iNOS. The sh-BMAL1 intervention reversed suvorexant's effects, suggesting that BMAL1 may play a role in how suvorexant influences cognitive impairment in CRSD rats. CONCLUSION:Suvorexant may improve CRSD-related cognitive impairment by modulating microglial activation and the BMAL1/NLRP3 pathway, providing potential insights into related conditions.

MicroRNA-146a-5p (miR-146a-5p) actively participates in the process of cerebral ischemia–reperfusion (CI/R) injury. Dysregulation of the tumor necrosis factor receptor-associated factor 6 (TRAF6)/nuclear factor kappa-B (NF-κB) p65 axis is closely associated with inflammatory response. This study aimed to investigate the potential involvement of miR-146a-5p and TRAF6/NF-κB p65 in mediating CI/R progression in vitro. HT-22 cells were challenged with oxygen–glucose deprivation/reoxygenation (OGD/R) to simulate CI/R in vitro. HT-22 cells were transfected with miR-146a-5p mimics or TRAF6 overexpression constructs. The impact of miR-146a-5p on apoptosis, inflammation, and TRAF6/NF-κB p65 activation were investigated. OGD/R inhibited HT-22 cell viability, induced apoptosis, reduced miR-146a-5p levels and activated the TRAF6/NF-κB p65 pathway. MiR-146a-5p mimics reduced pro-inflammatory factor release, limited apoptosis-related protein expression, and inactivated the TRAF6/NF-κB p65 pathway in OGD/R-challenged HT-22 cells. Mechanistically, miR-146a-5p was verified to bind to TRAF6 3’UTR. TRAF6 overexpression reversed the beneficial effects of miR-146a-5p mimics on apoptosis, inflammation, and TRAF6/NF-κB p65 activation. This work revealed that miR-146a-5p targeted TRAF6 and suppressed the TRAF6/NF-κB p65 pathway, thereby reducing OGD/R-induced inflammation and apoptosis in HT-22 cells. These findings suggest the potential of the miR-146a-5p/TRAF6/NF-κB p65 axis in the treatment of CI/R.

Ischemic stroke (IS) is known for its high morbidity, disability and mortality rates, and studies designed to explore its pathophysiological mechanisms and identify novel therapeutic strategies are urgently needed. We aimed to probe the effects of the deubiquitinase OTUD3-IRP2-p53/PTGS2 pathway on cerebral ischemia‒reperfusion (I/R) injury and hippocampal neuron ferroptosis. A cerebral I/R mouse model was established. Furthermore, lentiviral vectors that overexpressed OTUD3 and knocked down IRP2 were constructed, and a series of assays were performed to probe the OTUD3/IRP2/p53/PTGS2 mechanism. An oxygen‒glucose deprivation and reoxygenation (OGD/R) model of mouse hippocampal neurons was constructed. Then, OTUD3 and IRP2 were knocked down and overexpressed, and p53 was overexpressed to explore the mechanism of the OTUD3/IRP2/p53/PTGS2 pathway. OTUD3 and IRP2 were expressed at low levels in cerebral I/R models. OTUD3 promoted IRP2 expression to protect damaged hippocampal neurons. Moreover, IRP2 affected ferroptosis in hippocampal neurons. In addition, IRP2 inhibited p53. After IRP2 and p53 were overexpressed, IRP2 regulated the p53/PTGS2 pathway and affected ferroptosis in hippocampal neurons. In vivo, after overexpressing OTUD3 and knocking down IRP2, we found that overexpression of OTUD3 promoted IRP2 expression to reduce ferroptosis in hippocampal neurons and improve cerebral I/R injury via the inhibition of the p53/PTGS2 pathway. The deubiquitinase OTUD3 stabilized IRP2 expression to reduce hippocampal neuron ferroptosis via the p53/PTGS2 pathway to subsequently ameliorate cerebral I/R injury.

Sleep deprivation (SD) is a prevalent sleep issue in modern society that significantly impairs neurological function and quality of life in affected individuals. This study seeks to investigate the involvement of the miR‑155‑5p/BDNF axis in SD mice, aiming to establish a theoretical foundation for potential treatment strategies. Male C57BL/6 mice were utilized in the construction of a SD model using the flower pot technique. HT22 cells were selected for cellular experiments. The Morris water maze was employed to assess the learning and memory capabilities of the mice. HE staining was utilized to observe pathological changes in hippocampal tissue. Levels of IL‑1β, IL‑6, and TNF‑α were analyzed using ELISA. The expression level of miR‑155‑5p was quantified via RT‑qPCR. The binding between miR‑155‑5p and brain‑derived neurotrophic factor (BDNF) was confirmed through a dual‑luciferase reporter assay. Apoptosis of hippocampal neurons was assessed using TUNEL. Western blot analysis was conducted to evaluate the expression levels of BDNF, p65, and p‑p65. The Morris water maze test revealed that the mice exhibited prolonged escape latency, decreased swimming velocity, and reduced time spent in the target platform quadrant, which are indicative of a successful construction of the SD model. The observed cognitive deficits in the mice were associated with SD‑induced damage to the hippocampal tissue, leading to increased levels of miR‑155‑5p and decreased levels of BDNF. miR‑155‑5p was found to directly bind to BDNF, thereby suppressing its mRNA and protein expression. The upregulation of BDNF effectively mitigated hippocampal damage by attenuating cell apoptosis and reducing inflammation levels in SD mice. Additionally, the BDNF/NF‑κB pathway was found to be suppressed in SD mice through the downregulation of miR‑155‑5p. Therefore, the silencing of miR‑155‑5p inhibited the activation of the NF‑κB pathway by upregulating BDNF, which improved long‑term memory and reduced neuronal damage in SD mice.

OTU domain-containing protein 3 (OTUD3) is a crucial deubiquitinase that exhibits significant expression differences across various disease models. OTUD3 plays a role in regulating biological functions such as apoptosis, inflammatory responses, cell cycle, proliferation, and invasion in different cell types. By deubiquitinating key substrate proteins, OTUD3 is involved in essential physiological and pathological processes, including innate antiviral immunity, neural development, neurodegenerative diseases, and cancer. OTUD3 exhibits tumor-suppressive effects in breast cancer, esophageal cancer, colon cancer, and papillary thyroid cancer, but acts as an oncogenic in liver and lung cancers. OTUD3 serves as a biomarker in predicting diagnosing, and assessing prognosis for certain malignancies. Despite its potential, the molecular mechanisms of OTUD3 in many diseases are still not well-understood, and exploring OTUD3's regulatory mechanisms is essential for comprehending its roles in immunity and disease. Future research will focus on developing OTUD3-targeted therapies.

BACKGROUND:CI/R, characterized by ischemic injury following abrupt reestablishment of blood flow, can cause oxidative stress, mitochondrial dysfunction, and apoptosis. We used oxygen-glucose deprivation/reoxygenation (OGD/R) induced injury in HT22 and primary mouse cortical neurons (MCN) as a model for CI/R. OBJECTIVE:This study investigates the role of miR-188-5p in hippocampal neuron cell injury associated with Cerebral Ischemia-Reperfusion (CI/R). METHODS:HT22 and MCN cells were induced by OGD/R to construct an in vitro model of CI/R. Cell apoptosis and proliferation were assessed using flow cytometry and the Cell Counting Kit-8 (CCK8). ELISA was conducted to measure the levels of IL-1β, IL-6, and TNF-α. Moreover, the interaction between miR-188-5p and IL6ST was investigated using dual luciferase assay, the expression of miR-188-5p, Bax, cleaved-caspase3, IL-6, Bcl-2, IL-1β, TNF-α, IL6ST, NFκB, NLRP3 and STAT3 was evaluated using RT-qPCR or Western blot, and immunofluorescence was used to analyze the co-expression of p-STAT3 and NLRP3 in neuronal cells. RESULTS:OGD/R reduced proliferation and miR-188-5p levels and increased IL6ST expression, inflammation, and apoptosis in HT22 and MCN cells. Moreover, miR-188-5p was found to bind to IL6ST. Mimics of miR-188-5p reduced apoptosis, lowered the expression of cleaved-caspase3 and Bax proteins, and elevated Bcl-2 protein expression in cells treated with OGD/R. Overexpression of miR-188-5p decreased the levels of NLRP3 and p-STAT3 in the OGD/R group. Furthermore, the overexpression of miR-188-5p reduced IL6ST, p- NFκB/NFκB, p-STAT3/STAT3, and NLRP3 proteins in OGD/R, and these effects could be reversed by IL6ST overexpression. CONCLUSION:Mimics of miR-188-5p were found to inhibit inflammation and the STAT3/NLRP3 pathway via IL6ST, thereby ameliorating injury in HT22 and MCN cells treated with OGD/R in the context of CI/R.

Introduction: The purpose of this study was to investigate the effects of bone marrow mesenchymal stem cells (BMSCs) exosomal miR-345-3p and tumor necrosis factor receptorassociated factor 6 (TRAF6) on cerebral ischemia reperfusion (CIR) injury. Exosomes (Exos) derived from BMSCs were isolated and identified. PC12 (rat pheochromocytoma) cells were used to establish an oxygen and glucose deprivation/reoxygenation (OGD/R) model. Methods: Cell counting kit-8, TUNEL staining, lactate dehydrogenase staining, RT-qPCR, and western blotting were utilized for analyzing the functions of miR-345-3p about PC12 cells. Dualluciferase reporter experiment was then to confirm the link between miR-345-3p and TRAF6. Finally, using male SD rats, the middle cerebral artery occlusion (MCAO) model was constructed. Regulation of I/R damage in MCAO rats of miR-345-3p and TRAF6 were further explored in the changes of modified neurological severity score, cerebral infarction pictures, relative infarct volume, and histopathological changes. After OGD/R treatment, neuronal apoptosis was dramatically increased. After treatment with exosomal miR-345-3p, OGD/R-induced neuroapoptosis was dramatically inhibited. Exosomal miR-345-3p inhibited OGD/R-induced neuroapoptosis by downregulating the expression of TRAF6. However, the miR-345-3p inhibitor aggravated the changes caused by OGD/R. Results: The corresponding regulations of miR-345-3p were reversed with TRAF6 overexpression. The animal experiments in vivo further verified that miR-345-3p ameliorated brain I/R injury in MCAO rats by targeting TRAF6. Conclusion: This study found that BMSCs-exosomal miR-345-3p protected against CIR injury by decreasing TRAF6.

Objective To investigate the therapeutic effect of liraglutide on acute cerebral infarction(ACI)in patients with type 2 diabetes mellitus(T2DM),and its effect on neurological function and serum vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(bFGF).Methods A total of 160 patients with ACI accompanied by T2DM admitted to Haikou People's Hospital from March 2020 to March 2022 were randomly divided into observation group and control group(n=80,each).Both groups of patients were given routine treatment for ACI.On this basis,the control group was treated with insulin aspart and insulin glargine,while the observation group was treated with liraglutide and insulin glargine.Both groups received continuous treatment for 3 months.In addition to clinical efficacy,fasting blood glucose(FBG)before and after treatment,2-hour postprandial blood glucose(2hPG),hemoglobin A1e(HbA1c),insulin resistance index(HOMA-IR),National Institutes of Health Stroke Scale(NIHSS),and modified Rankin Scale(mRS)scores of two groups of patients was compared.Enzyme linked immunosorbent assay was used to detect the levels of serum VEGF and bFGF before treatment and after 1 and 3 months of treatment in both groups.Results After treatment,the levels of FBG,bFGF,HbA1c,HOMA-IR,and the scores of NIHSS and mRS in the observation group decreased compared to those before treatment,and the above indicators levels in the observation group were significantly lower than those in the control group(P＜0.05).The total clinical effective rate of the observation group was significantly higher than that of the control group[95.00％(76/80)vs 83.75％(67/80),x2=5.331,P=0.021].After treatment,the levels of serum VEGF and bFGF showed a gradual downward trend in both groups(P＜0.05).After 1 month and 3 months of treatment,the serum VEGF and bFGF levels in the observation group were significantly higher than those in the control group(P＜0.05).Conclusion Liraglutide combined with insulin glargine can effectively control blood sugar,reduce insulin resistance,alleviate neurological damage,upregulate serum VEGF and bFGF levels,and improve clinical efficacy in T2DM patients with ACI.