Naodesheng Pills Ameliorate Cerebral Ischemia Reperfusion-Induced Ferroptosis via Inhibition of the ERK1/2 Signaling Pathway.

Background: Ferroptosis plays a crucial role in the occurrence and development of cerebral ischemia-reperfusion (I/R) injury and is regulated by mitogen-activated protein kinase 1/2 (ERK1/2). In China, Naodesheng Pills (NDSP) are prescribed to prevent and treat cerebrosclerosis and stroke. However, the protective effects and mechanism of action of NDSP against cerebral I/R-induced ferroptosis remain unclear. We investigated whether NDSP exerts its protective effects against I/R injury by regulating ferroptosis and aimed to elucidate the underlying mechanisms. Methods: The efficacy of NDSP was evaluated using a Sprague-Dawley rat model of middle cerebral artery occlusion and an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model. Brain injury was assessed using 2,3,5-triphenyltetrazolium chloride (TTC), hematoxylin and eosin staining, Nissl staining, and neurological scoring. Western blotting was performed to determine the expression levels of glutathione peroxidase 4 (GPX4), divalent metal-ion transporter-1 (DMT1), solute carrier family 7 member 11 (SLC7A11), and transferrin receptor 1 (TFR1). Iron levels, oxidative stress, and mitochondrial morphology were also evaluated. Network pharmacology was used to assess the associated mechanisms. Results: NDSP (1.08 g/kg) significantly improved cerebral infarct area, cerebral water content, neurological scores, and cerebral tissue damage. Furthermore, NDSP inhibited I/R- and OGD/R-induced ferroptosis, as evidenced by the increased protein expression of GPX4 and SLC7A11, suppression of TFR1 and DMT1, and an overall reduction in oxidative stress and Fe2+ levels. The protective effects of NDSP in vitro were abolished by the GPX4 inhibitor RSL3. Network pharmacology analysis revealed that ERK1/2 was the core target gene and that NDSP reduced the amount of phosphorylated ERK1/2. Conclusion: NDSP exerts its protective effects against I/R by inhibiting cerebral I/R-induced ferroptosis, and this mechanism is associated with the regulation of ferroptosis via the ERK1/2 signaling pathway.

Ketamine-induced prevention of SD-associated late infarct progression in experimental ischemia.

Spreading depolarizations (SDs) occur frequently in patients with malignant hemispheric stroke. In animal-based experiments, SDs have been shown to cause secondary neuronal damage and infarct expansion during the initial period of infarct progression. In contrast, the influence of SDs during the delayed period is not well characterized yet. Here, we analyzed the impact of SDs in the delayed phase after cerebral ischemia and the potential protective effect of ketamine. Focal ischemia was induced by distal occlusion of the left middle cerebral artery in C57BL6/J mice. 24 h after occlusion, SDs were measured using electrocorticography and laser-speckle imaging in three different study groups: control group without SD induction, SD induction with potassium chloride, and SD induction with potassium chloride and ketamine administration. Infarct progression was evaluated by sequential MRI scans. 24 h after occlusion, we observed spontaneous SDs with a rate of 0.33 SDs/hour which increased during potassium chloride application (3.37 SDs/hour). The analysis of the neurovascular coupling revealed prolonged hypoemic and hyperemic responses in this group. Stroke volume increased even 24 h after stroke onset in the SD-group. Ketamine treatment caused a lesser pronounced hypoemic response and prevented infarct growth in the delayed phase after experimental ischemia. Induction of SDs with potassium chloride was significantly associated with stroke progression even 24 h after stroke onset. Therefore, SD might be a significant contributor to delayed stroke progression. Ketamine might be a possible drug to prevent SD-induced delayed stroke progression.

GABAergic system and chloride cotransporters as potential therapeutic targets to mitigate cell death in ischemia.

Gamma aminobutyric acid (GABA) is a critical inhibitory neurotransmitter in the central nervous system that plays a vital role in modulating neuronal excitability. Dysregulation of GABAergic signaling, particularly involving the cotransporters NKCC1 and KCC2, has been implicated in various pathologies, including epilepsy, schizophrenia, autism spectrum disorder, Down syndrome, and ischemia. NKCC1 facilitates chloride influx, whereas KCC2 mediates chloride efflux via potassium gradient. Altered expression and function of these cotransporters have been associated with excitotoxicity, inflammation, and cellular death in ischemic events characterized by reduced cerebral blood flow, leading to compromised tissue metabolism and subsequent cell death. NKCC1 inhibition has emerged as a potential therapeutic approach to attenuate intracellular chloride accumulation and mitigate neuronal damage during ischemic events. Similarly, targeting KCC2, which regulates chloride efflux, holds promise for improving outcomes and reducing neuronal damage under ischemic conditions. This review emphasizes the critical roles of GABA, NKCC1, and KCC2 in ischemic pathologies and their potential as therapeutic targets. Inhibiting or modulating the activity of these cotransporters represents a promising strategy for reducing neuronal damage, preventing excitotoxicity, and improving neurological outcomes following ischemic events. Furthermore, exploring the interactions between natural compounds and NKCC1/KCC2 provides additional avenues for potential therapeutic interventions for ischemic injury.

Polyphyllin I alleviates neuroinflammation after cerebral ischemia-reperfusion injury via facilitating autophagy-mediated M2 microglial polarization.

Microglial activation and polarization play a central role in poststroke inflammation and neuronal damage. Modulating microglial polarization from pro-inflammatory to anti-inflammatory phenotype is a promising therapeutic strategy for the treatment of cerebral ischemia. Polyphyllin I (PPI), a steroidal saponin, shows multiple bioactivities in various diseases, but the potential function of PPI in cerebral ischemia is not elucidated yet. In our study, the influence of PPI on cerebral ischemia-reperfusion injury was evaluated. Mouse middle cerebral artery occlusion (MCAO) model and oxygen-glucose deprivation and reoxygenation (OGD/R) model were constructed to mimic cerebral ischemia-reperfusion injury in vivo and in vitro. TTC staining, TUNEL staining, RT-qPCR, ELISA, flow cytometry, western blot, immunofluorescence, hanging wire test, rotarod test and foot-fault test, open-field test and Morris water maze test were performed in our study. We found that PPI alleviated cerebral ischemia-reperfusion injury and neuroinflammation, and improved functional recovery of mice after MCAO. PPI modulated microglial polarization towards anti-inflammatory M2 phenotype in MCAO mice in vivo and post OGD/R in vitro. Besides, PPI promoted autophagy via suppressing Akt/mTOR signaling in microglia, while inhibition of autophagy abrogated the effect of PPI on M2 microglial polarization after OGD/R. Furthermore, PPI facilitated autophagy-mediated ROS clearance to inhibit NLRP3 inflammasome activation in microglia, and NLRP3 inflammasome reactivation by nigericin abolished the effect of PPI on M2 microglia polarization. In conclusion, PPI alleviated post-stroke neuroinflammation and tissue damage via increasing autophagy-mediated M2 microglial polarization. Our data suggested that PPI had potential for ischemic stroke treatment.

Utilizing network pharmacology, molecular docking, and animal models to explore the therapeutic potential of the WenYang FuYuan recipe for cerebral ischemia-reperfusion injury through AGE-RAGE and NF-κB/p38MAPK signaling pathway modulation.

BACKGROUND: Stroke is a debilitating condition with high morbidity, disability, and mortality that significantly affects the quality of life of patients. In China, the WenYang FuYuan recipe is widely used to treat ischemic stroke. However, the underlying mechanism remains unknown, so exploring the potential mechanism of action of this formula is of great practical significance for stroke treatment. OBJECTIVE: This study employed network pharmacology, molecular docking, and in vivo experiments to clarify the active ingredients, potential targets, and molecular mechanisms of the WenYang FuYuan recipe in cerebral ischemia-reperfusion injury, with a view to providing a solid scientific foundation for the subsequent study of this recipe. MATERIALS AND METHODS: Active ingredients of the WenYang FuYuan recipe were screened using the traditional Chinese medicine systems pharmacology database and analysis platform. Network pharmacology approaches were used to explore the potential targets and mechanisms of action of the WenYang FuYuan recipe for the treatment of cerebral ischemia-reperfusion injury. The Middle Cerebral Artery Occlusion/Reperfusion 2 h Sprague Dawley rat model was prepared, and TTC staining and modified neurological severity score were applied to examine the neurological deficits in rats. HE staining and Nissl staining were applied to examine the pathological changes in rats. Immunofluorescence labeling and Elisa assay were applied to examine the expression levels of certain proteins and associated factors, while qRT-PCR and Western blotting were applied to examine the expression levels of linked proteins and mRNAs in disease-related signaling pathways. RESULTS: We identified 62 key active ingredients in the WenYang FuYuan recipe, with 222 highly significant I/R targets, forming 138 pairs of medication components and component-targets, with the top five being Quercetin, Kaempferol, Luteolin, ß-sitosterol, and Stigmasterol. The key targets included TP53, RELA, TNF, STAT1, and MAPK14 (p38MAPK). Targets related to cerebral ischemia-reperfusion injury were enriched in chemical responses, enzyme binding, endomembrane system, while enriched pathways included lipid and atherosclerosis, fluid shear stress and atherosclerosis, AGE-RAGE signaling in diabetic complications. In addition, the main five active ingredients and targets in the WenYang FuYuan recipe showed high binding affinity (e.g. Stigmasterol and MAPK14, total energy <-10.5 Kcal/mol). In animal experiments, the WenYang FuYuan recipe reduced brain tissue damage, increased the number of surviving neurons, and down-regulated S100ß and RAGE protein expression. Moreover, the relative expression levels of key targets such as TP53, RELA and p38MAPK mRNA were significantly down-regulated in the WenYang FuYuan recipe group, and serum IL-6 and TNF-a factor levels were reduced. After WenYang FuYuan recipe treatment, the AGE-RAGE signaling pathway and downstream NF-kB/p38MAPK signaling pathway-related proteins were significantly modulated. CONCLUSION: This study utilized network pharmacology, molecular docking, and animal experiments to identify the potential mechanism of the WenYang FuYuan recipe, which may be associated with the regulation of the AGE-RAGE signaling pathway and the inhibition of target proteins and mRNAs in the downstream NF-kB/p38MAPK pathway.

Research progress of prodrugs for the treatment of cerebral ischemia.

It is well-known that pharmacotherapy plays a pivotal role in the treatment and prevention of cerebral ischemia. Nevertheless, existing drugs, including numerous natural products, encounter various challenges when applied in cerebral ischemia treatment. These challenges comprise poor brain absorption due to low blood-brain barrier (BBB) permeability, limited water solubility, inadequate bioavailability, poor stability, and rapid metabolism. To address these issues, researchers have turned to prodrug strategies, aiming to mitigate or eliminate the adverse properties of parent drug molecules. In vivo metabolism or enzymatic reactions convert prodrugs into active parent drugs, thereby augmenting BBB permeability, improving bioavailability and stability, and reducing toxicity to normal tissues, ultimately aiming to enhance treatment efficacy and safety. This comprehensive review delves into multiple effective prodrug strategies, providing a detailed description of representative prodrugs developed over the past two decades. It underscores the potential of prodrug approaches to improve the therapeutic outcomes of currently available drugs for cerebral ischemia. The publication of this review serves to enrich current research progress on prodrug strategies for the treatment and prevention of cerebral ischemia. Furthermore, it seeks to offer valuable insights for pharmaceutical chemists in this field, offer guidance for the development of drugs for cerebral ischemia, and provide patients with safer and more effective drug treatment options.

A study on the mechanism of Beclin-1 m6A modification mediated by catalpol in protection against neuronal injury and autophagy following cerebral ischemia.

OBJECTIVE: Catalpol (CAT) has various pharmacological activities and plays a protective role in cerebral ischemia. It has been reported that CAT played a protective role in cerebral ischemia by upregulaing NRF1 expression. Bioinformatics analysis reveals that NRF1 can be used as a transcription factor to bind to the histone acetyltransferase KAT2A. However, the role of KAT2A in cerebral ischemia remains to be studied. Therefore, we aimed to investigate the role of CAT in cerebral ischemia and its related mechanism. METHODS: In vitro, a cell model of oxygen and glucose deprivation/reperfusion (OGD/R) was constructed, followed by evaluation of neuronal injury and the expression of METTL3, Beclin-1, NRF1, and KAT2A. In vivo, a MCAO rat model was prepared by means of focal cerebral ischemia, followed by assessment of neurological deficit and brain injury in MCAO rats. Neuronal autophagy was evaluated by observation of autophagosomes in neurons or brain tissues by TEM and detection of the expression of LC3 and p62. RESULTS: In vivo, CAT reduced the neurological function deficit and infarct volume, inhibited neuronal apoptosis in the cerebral cortex, and significantly improved neuronal injury and excessive autophagy in MCAO rats. In vitro, CAT restored OGD/R-inhibited cell viability, inhibited cell apoptosis, LDH release, and neuronal autophagy. Mechanistically, CAT upregulated NRF1, NRF1 activated METTL3 via KAT2A transcription, and METTL3 inhibited Beclin-1 via m6A modification. CONCLUSION: CAT activated the NRF1/KAT2A/METTL3 axis and downregulated Beclin-1 expression, thus relieving neuronal injury and excessive autophagy after cerebral ischemia.

Integrated pharmacokinetic-pharmacodynamic modeling and metabolomic research on polyphenol-rich fraction of Thymus quinquecostatus Celak. Alleviating cerebral ischemia-reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Thymus quinquecostatus Celak., a member of thymus genus in Lamiaceae family, has been used as a folk medicine for relieving exterior syndrome and alleviating pain in China. The polyphenol-rich fraction (PRF) derived from Thymus quinquecostatus Celak. had been validated that it can protect cerebral ischemia-reperfusion injury (CIRI) by activating Keap1/Nrf2/HO-1 signaling pathway. AIM OF THIS STUDY: To explore effective components and their pharmacokinetic and pharmacodynamic characteristics as well as possible mechanisms of PRF in treating CIRI. MATERIALS AND METHODS: Normal treated group (NTG) and tMCAO model treated group (MTG) rats were administrated PRF intragastrically. The prototype components and metabolites of PRF in plasma and brain were analyzed by the UPLC-Q-Exactive Orbitrap MSn method. Subsequently, the pharmacokinetics properties of indicative components were performed based on HPLC-QQQ-MS/MS. SOD and LDH activities were determined to study the pharmacodynamic (PD) properties of PRF. The PK-PD relationship of PRF was constructed. In addition, the effect of PRF on endogenous metabolites in plasma and brain was investigated using metabolomic method. RESULTS: Salvianic acid A, caffeic acid, rosmarinic acid, scutellarin, and apigenin-7-O-glucuronide were selected as indicative components based on metabolic analysis. The non-compartmental parameters were calculated for indicative components in plasma and brain of NTG and MTG rats. Furthermore, single-component and multi-component PK-PD modeling involved Emax, Imax PD models for effect indexes were fitted as well as ANN models were established, which indicated that these components can work together to regulate SOD and LDH activities in plasma and SOD activity in brain tissue to improve CIRI. Additionally, PRF may ameliorate CIRI by regulating the disorder of endogenous metabolites in lipid metabolism, amino acid metabolism, and purine metabolism pathways in vivo, among which lipid metabolism and purine metabolism are closely related to oxidative stress. CONCLUSION: The PK-PD properties of effect substances and mechanisms of PRF anti-CIRI were further elaborated. The findings provide a convincing foundation for the application of T. quinquecostatus Celak. in the maintenance of human health disorders.

Cholestane-3ß,5α,6ß-Triol Inhibits Acid-Sensing Ion Channels and Reduces Acidosis-Mediated Ischemic Brain Injury.

BACKGROUND: Activation of the acid-sensing ion channels (ASICs) by tissue acidosis, a common feature of brain ischemia, contributes to ischemic brain injury, while blockade of ASICs results in protection. Cholestane-3ß,5α,6ß-triol (Triol), a major cholesterol metabolite, has been demonstrated as an endogenous neuroprotectant; however, the mechanism underlying its neuroprotective activity remains elusive. In this study, we tested the hypothesis that inhibition of ASICs is a potential mechanism. METHODS: The whole-cell patch-clamp technique was used to examine the effect of Triol on ASICs heterogeneously expressed in Chinese hamster ovary cells and ASICs endogenously expressed in primary cultured mouse cortical neurons. Acid-induced injury of cultured mouse cortical neurons and middle cerebral artery occlusion-induced ischemic brain injury in wild-type and ASIC1 and ASIC2 knockout mice were studied to examine the protective effect of Triol. RESULTS: Triol inhibits ASICs in a subunit-dependent manner. In Chinese hamster ovary cells, it inhibits homomeric ASIC1a and ASIC3 without affecting ASIC1ß and ASIC2a. In cultured mouse cortical neurons, it inhibits homomeric ASIC1a and heteromeric ASIC1a-containing channels. The inhibition is use-dependent but voltage- and pH-independent. Structure-activity relationship analysis suggests that hydroxyls at the 5 and 6 positions of the A/B ring are critical functional groups. Triol alleviates acidosis-mediated injury of cultured mouse cortical neurons and protects against middle cerebral artery occlusion-induced brain injury in an ASIC1a-dependent manner. CONCLUSIONS: Our study identifies Triol as a novel ASIC inhibitor, which may serve as a new pharmacological tool for studying ASICs and may also be developed as a potential drug for treating stroke.

Triptolide alleviates cerebral ischemia/reperfusion injury via regulating the Fractalkine/CX3CR1 signaling pathway.

PURPOSE: To evaluate the potential efficacy of Triptolide (TP) on cerebral ischemia/reperfusion injury (CIRI) and to uncover the underlying mechanism through which TP regulates CIRI. METHODS: We constructed a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model to simulate CIRI, and established a lipopolysaccharide (LPS)-stimulated BV-2 cell model to mimic the inflammatory state during CIRI. The neurological deficits score (NS) of mice were measured for assessment of neurologic functions. Both the severity of cerebral infarction and the apoptosis level in mouse brain tissues or cells were respectively evaluated using corresponding techniques. The expression levels of Ionized calcium binding adapter molecule 1 (IBA-1), Inductible Nitric Oxide Synthase (iNOS), Arginase 1 (Arg-1), Tumor necrosis factor-α (TNF-α), Interleukin 1ß (IL-1ß), Cysteine histoproteinase S (CTSS), Fractalkine, chemokine C-X3-C motif receptor 1 (CX3CR1), BCL-2-associated X protein (BAX), and antiapoptotic proteins (Bcl-2) were detected using immunofluorescence, qRT-PCR as well as Western blot, respectively. RESULTS: Relative to the Sham group, treatment with TP attenuated the increased NS, infarct area and apoptosis levels observed in MCAO/R mice. Upregulated expression levels of IBA-1, iNOS, Arg-1, TNF-α and IL-1ß were found in MCAO/R mice, while TP suppressed iNOS, TNF-α and IL-1ß expression, and enhanced Arg-1 expression in both MCAO/R mice and LPS-stimulated BV-2 cells. Besides, TP inhibited the CTSS/Fractalkine/CX3CR1 pathway activation in both MCAO/R mice and LPS-induced BV-2 cells, while overexpression of CTSS reversed such effect. Co-culturing HT-22 cells with TP+LPS-treated BV-2 cells led to enhanced cell viability and decreased apoptosis levels. However, overexpression of CTSS further aggravated HT-22 cell injury. CONCLUSION: TP inhibits not only microglia polarization towards the M1 phenotype by suppressing the CTSS/Fractalkine/CX3CR1 pathway activation, but also HT-22 apoptosis by crosstalk with BV-2 cells, thereby ameliorating CIRI. These findings reveal a novel mechanism of TP in improving CIRI, and offer potential implications for addressing the preventive and therapeutic strategies of CIRI.

Remodeling brain pathological microenvironment to lessen cerebral ischemia injury by multifunctional injectable hydrogels.

Ischemia stroke is one of the leading causes of death and disability worldwide. Owing to the limited delivery efficiency to the brain caused by the blood-brain barrier (BBB) and off-target effects of systemic treatment, it is crucial to develop an in situ drug delivery system to improve the therapeutic effect in ischemic stroke. Briefly, we report a multifunctional in situ hydrogel delivery system for the co-delivery of reactive oxygen species (ROS)-responsive nanoparticles loaded with atorvastatin calcium (DSPE-se-se-PEG@AC NPs) and ß-nerve growth factor (NGF), which is expected to remodel pathological microenvironment for improving cerebral ischemia injury. The in vitro results exhibited the multifunctional hydrogel scavenged oxygen-glucose deprivation (OGD)-induced free radical, rescued the mitochondrial function, and maintained the survival and function of neurons, hence reducing neuronal apoptosis and neuroinflammation, consequently relieving ischemia injury in hippocampal neurons cell line (HT22). In the rat ischemia stroke model, the hydrogel significantly minified cerebral infarction by regulating inflammatory response, saving apoptotic neurons, and promoting angiogenesis and neurogenesis. Besides, the hydrogel distinctly improved the rats' neurological deficits after cerebral ischemia injury over the long-term observation. In conclusion, the in-situ hydrogel platform has demonstrated promising therapeutic effects in both in vitro and in vivo studies, indicating its potential as a new and effective therapy.

LC-MS based metabonomics study on protective mechanism of ESWW in cerebral ischemia via CYTC/Apaf-1/NDRG4 pathway.

BACKGROUND: Ershiwuwei Zhenzhu pills was originally recorded in the Tibetan medical book Si Bu Yi Dian in the 8th century AD and is now included in the Pharmacopoeia of the People's Republic of China (2020). The pills can calm the nerves and open the mind as well as treat cerebral ischemia reperfusion injury, stroke, hemiplegia. However, its quality standards have not yet been established, and the therapeutic effect on cerebral ischemia by regulating the mitochondrial apoptosis pathway has not been elucidated. STUDY DESIGN AND METHODS: LC-MS was used to establish quality standards for Ershiwuwei Zhenzhu pills. Metabonomics, molecular docking, neuroethology, cerebral infarction ratio, pathological detection of diencephalon, cortex, and hippocampus, and molecular biology techniques were used to reveal the mechanism of the pills in regulating the mitochondrial apoptosis pathway to treat cerebral ischemia. RESULTS: The contents of 20 chemical components in Ershiwuwei Zhenzhu pills from 12 batches and 8 manufacturers was determined for the first time. Eleven differential metabolites and three metabolic pathways, namely, fructose and mannose metabolism, glycerophospholipid metabolism, and purine metabolism, were identified by metabonomics. The pills improved the neuroethology abnormalities of MCAO rats and the pathological damage in the diencephalon and decreased the ratio of cerebral infarction. It also significantly reduced the mRNA expression of AIF, Apaf-1, cleared caspase8, CytC, and P53 mRNA in the brain tissue and the protein expression of Apaf-1 and CYTC and increased the protein expression of NDRG4. CONCLUSION: In vitro quantitative analysis of the in vitro chemical components of Ershiwuwei Zhenzhu pills has laid the foundation for improving its quality control. The potential mechanism of the pills in treating cerebral ischemia may be related to the Apaf-1/CYTC/NDRG4 apoptosis pathway. This work provides guidance for clinical drug use for patients.

The effect of calpain inhibitor-I on copper oxide nanoparticle-induced damage and cerebral ischemia-reperfusion in a rat model.

This study aimed to investigate the effects of the calpain inhibitor N-Acetyl-Leu-Leu-norleucinal (ALLN) on neuroapoptotic cell damage caused by Copper Oxide Nanoparticles (CuO-NP) and exacerbation of damage through brain ischemia/reperfusion (I/R) in a rat model. Male Wistar Albino rats (n=80) were divided into eight groups: Control, I/R, CuO-NP, CuO-NP+I/R, I/R+ALLN, CuO-NP+ALLN, CuO-NP+I/R+ALLN, and DMSO. Biochemical markers (MBP, S100B, NEFL, NSE, BCL-2, Cyt-C, Calpain, TNF-α, Caspase-3, MDA, and CAT) were measured in serum and brain tissue samples. Histological examinations (H&E staining), DNA fragmentation analysis (TUNEL) were performed, along with Caspase-3 assessment. The ALLN-treated groups exhibited significant improvements in biochemical markers and a remarkable reduction in apoptosis compared to the damaged groups (CuO-NP and I/R). H&E and Caspase-3 staining revealed damage-related morphological changes and reduced apoptosis in the ALLN-treated group. However, no differences were observed among the groups with TUNEL staining. The findings suggest that ALLN, as a calpain inhibitor, has potential implications for anti-apoptotic treatment, specifically in mitigating neuroapoptotic cell damage caused by CuO-NP and I/R.

N6022 attenuates cerebral ischemia/reperfusion injury-induced microglia ferroptosis by promoting Nrf2 nuclear translocation and inhibiting the GSNOR/GSTP1 axis.

Stroke poses a significant risk of mortality, particularly among the elderly population. The pathophysiological process of ischemic stroke is complex, and it is crucial to elucidate its molecular mechanisms and explore potential protective drugs. Ferroptosis, a newly recognized form of programmed cell death distinct from necrosis, apoptosis, and autophagy, is closely associated with the pathophysiology of ischemic stroke. N6022, a selective inhibitor of S-nitrosoglutathione reductase (GSNOR), is a "first-in-class" drug for asthma with potential therapeutic applications. However, it remains unclear whether N6022 exerts protective effects in ischemic stroke, and the precise mechanisms of its action are unknown. This study aimed to investigate whether N6022 mitigates cerebral ischemia/reperfusion (I/R) injury by reducing ferroptosis and to elucidate the underlying mechanisms. Accordingly, we established an oxygen-glucose deprivation/reperfusion (OGD/R) cell model and a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model to mimic cerebral I/R injury. Our data, both in vitro and in vivo, demonstrated that N6022 effectively protected against I/R-induced brain damage and neurological deficits in mice, as well as OGD/R-induced BV2 cell damage. Mechanistically, N6022 promoted Nrf2 nuclear translocation, enhancing intracellular antioxidant capacity of SLC7A11-GPX4 system. Furthermore, N6022 interfered with the interaction of GSNOR with GSTP1, thereby boosting the antioxidant capacity of GSTP1 and attenuating ferroptosis. These findings provide novel insights, showing that N6022 attenuates microglial ferroptosis induced by cerebral I/R injury through the promotion of Nrf2 nuclear translocation and inhibition of the GSNOR/GSTP1 axis.

Salidroside promotes angiogenesis after cerebral ischemia in mice through Shh signaling pathway.

AIMS: The purpose of this study was to explore the impacts of salidroside on vascular regeneration, vascular structural changes and long-term neurological recuperation following cerebral ischemia and its possible mechanism. MAIN METHODS: From Day 1 to Day 28, young male mice with middle cerebral artery blockage received daily doses of salidroside and measured neurological deficits. On the 7th day after stroke, the volume of cerebral infarction was determined using TTC and HE staining. Microvascular density, astrocyte coverage, angiogenesis and the expression of the Shh signaling pathway were detected by IF, qRTPCR and WB at 7, 14 and 28 days after stroke. Changes in blood flow, blood vessel density and diameter from stroke to 28 days were measured by the LSCI and TPMI. KEY FINDINGS: Compared with the dMACO group, the salidroside treatment group significantly promoted the recovery of neurological function. Salidroside was found to enhance cerebral blood flow perfusion and reduce the infarct on the 7th day after stroke. From the 7th to the 28th day after stroke, salidroside treatment boosted the expression of CD31, CD31+/BrdU+, and GFAP in the cortex around the infarction site. On the 14th day after stroke, salidroside significantly enhanced the width and density of blood vessels. Salidroside increased the expression of histones and genes in the Shh signaling pathway during treatment, and this effect was weakened by the Shh inhibitor Cyclopamine. SIGNIFICANCE: Salidroside can restore nerve function, improve cerebral blood flow, reduce cerebral infarction volume, increase microvessel density and promote angiogenesis via the Shh signaling pathway.

Sulfated Polysaccharide-Based Nanocarrier Drives Microenvironment-Mediated Cerebral Neurovascular Remodeling for Ischemic Stroke Treatment.

Stroke is a leading cause of global mortality and severe disability. However, current strategies used for treating ischemic stroke lack specific targeting capabilities, exhibit poor immune escape ability, and have limited drug release control. Herein, we developed an ROS-responsive nanocarrier for targeted delivery of the neuroprotective agent rapamycin (RAPA) to mitigate ischemic brain damage. The nanocarrier consisted of a sulfated chitosan (SCS) polymer core modified with a ROS-responsive boronic ester enveloped by a red blood cell membrane shell incorporating a stroke homing peptide. When encountering high levels of intracellular ROS in ischemic brain tissues, the release of SCS combined with RAPA from nanoparticle disintegration facilitates effective microglia polarization and, in turn, maintains blood-brain barrier integrity, reduces cerebral infarction, and promotes cerebral neurovascular remodeling in a mouse stroke model involving transient middle cerebral artery occlusion (tMCAO). This work offers a promising strategy to treat ischemic stroke therapy.

AZD1390, an ataxia telangiectasia mutated inhibitor, attenuates microglia-mediated neuroinflammation and ischemic brain injury.

AIMS: Excessive neuroinflammation mediated mainly by microglia plays a crucial role in ischemic stroke. AZD1390, an ataxia telangiectasia mutated (ATM) specific inhibitor, has been shown to promote radio-sensitization and survival in central nervous system malignancies, while the role of AZD1390 in ischemic stroke remains unknown. METHODS: Real-time PCR, western blot, immunofluorescence staining, flow cytometry and enzyme-linked immunosorbent assays were used to assess the activation of microglia and the release of inflammatory cytokines. Behavioral tests were performed to measure neurological deficits. 2,3,5-Triphenyltetrazolium chloride staining was conducted to assess the infarct volume. The activation of NF-κB signaling pathway was explored through immunofluorescence staining, western blot, co-immunoprecipitation and proximity ligation assay. RESULTS: The level of pro-inflammation cytokines and activation of NF-κB signaling pathway was suppressed by AZD1390 in vitro and in vivo. The behavior deficits and infarct size were partially restored with AZD1390 treatment in experimental stroke. AZD1390 restrict ubiquitylation and sumoylation of the essential regulatory subunit of NF-κB (NEMO) in an ATM-dependent and ATM-independent way respectively, which reduced the activation of the NF-κB pathway. CONCLUSION: AZD1390 suppressed NF-κB signaling pathway to alleviate ischemic brain injury in experimental stroke, and attenuated microglia activation and neuroinflammation, which indicated that AZD1390 might be an attractive agent for the treatment of ischemic stroke.

Advanced Nano-Drug Delivery Systems in the Treatment of Ischemic Stroke.

In recent years, the frequency of strokes has been on the rise year by year and has become the second leading cause of death around the world, which is characterized by a high mortality rate, high recurrence rate, and high disability rate. Ischemic strokes account for a large percentage of strokes. A reperfusion injury in ischemic strokes is a complex cascade of oxidative stress, neuroinflammation, immune infiltration, and mitochondrial damage. Conventional treatments are ineffective, and the presence of the blood-brain barrier (BBB) leads to inefficient drug delivery utilization, so researchers are turning their attention to nano-drug delivery systems. Functionalized nano-drug delivery systems have been widely studied and applied to the study of cerebral ischemic diseases due to their favorable biocompatibility, high efficiency, strong specificity, and specific targeting ability. In this paper, we briefly describe the pathological process of reperfusion injuries in strokes and focus on the therapeutic research progress of nano-drug delivery systems in ischemic strokes, aiming to provide certain references to understand the progress of research on nano-drug delivery systems (NDDSs).

Isobavachalcone alleviates ischemic stroke by suppressing HDAC1 expression and improving M2 polarization.

Ischemic stroke is a serious cerebrovascular condition. Isobavachalcone (ISO) has been documented to exhibit an anti-inflammatory effect across a variety of diseases; however, its protective impact on ischemic stroke remains unexplored. In this study, we evaluated the influence of ISO in both transient middle cerebral artery occlusion/reperfusion (tMCAO/R) rat models and oxygen-glucose deprivation/reperfusion (OGD/R) cell models. We observed that pretreatment with 50 mg/kg ISO diminished the volume of brain infarction, reduced brain edema, and ameliorated neurological deficits in rats. A reduction in Nissl bodies was noted in the tMCAO/R group, which was reversed following treatment with 50 mg/kg ISO. TUNEL/NeuN double staining revealed a decrease in TUNEL-positive cells in tMCAO/R rats treated with ISO. Furthermore, ISO treatment suppressed the expression of cleaved caspase-3 and BAX, while elevating the expression of BCL-2 in tMCAO/R rats. The levels of CD86 and iNOS were elevated in tMCAO/R rats; conversely, ISO treatment enhanced the expression of CD206 and Arg-1. Additionally, the expression of TNF-α, IL-6, and IL-1ß was elevated in tMCAO/R rats, whereas ISO treatment counteracted this effect. ISO treatment also increased the expression of TGF-ß and IL-10 in the ischemic penumbra of tMCAO/R rats. It was found that ISO treatment hindered microglial M1 polarization and favored M2 polarization. Histone Deacetylase 1 (HDAC1) is the downstream target protein of ISO, with ISO treatment resulting in decreased HDAC1 expression in both tMCAO/R rats and OGD/R-induced cells. Overexpression of HDAC1 was shown to promote microglial M1 polarization and inhibit M2 polarization in OGD/R+ISO cells. Overall, ISO treatment mitigated brain damage following ischemic stroke by promoting M2 polarization and attenuated ischemic injury by repressing HDAC1 expression.