Oxygen-glucose Deprivation/reperfusion Research Articles

Zerumbone-mediated post-ischemic neuroprotection: Reduction of ferroptosis through TFR1 downregulation in vitro.

Emerging studies have identified ferroptosis as a promising therapeutic target, the inhibition of which is hypothesized to mitigate brain injury and subsequent neuronal death following stroke. Zerumbone, a phytochemical sesquiterpene isolated from Zingiber zerumbet Smith, exhibit diverse therapeutic properties across a range of neurological disorders. This study aimed to elucidate the postischemic neuroprotective effects and regulatory impact of zerumbone on ferroptosis-mediated cell death following oxygen‒glucose deprivation/reperfusion (OGD/R) injury. We employed an in vitro OGD/R SH-SY5Y cell model of stroke to evaluate the postischemic neuroprotective effects of zerumbone, a lead molecule identified through literature studies. Moreover, assays were performed to assess how zerumbone affects lipid peroxide levels, intracellular reactive oxygen species (ROS), and mitochondrial membrane integrity. Furthermore, molecular docking simulations were carried out to determine the targets, and western blotting was performed to examine TFR1 protein expression. Zerumbone (0.5 µM) treatment at 1-hour postischemia increased cell viability (72.11 ± 0.98) and mitigated OGD/R-induced ischemic injury. Zerumbone significantly decreased intracellular ROS levels and lipid peroxide production while increasing mitochondrial membrane integrity, suggesting that zerumbone ameliorated OGD/R-induced ischemic injury by inhibiting ferroptosis in vitro. This finding was corroborated by our western blot analysis, which revealed that the antiferroptotic role of zerumbone was distinctly mediated through the downregulation of transferrin receptor 1 (TFR1) protein expression. This communication, for the first time, highlights the feasibility of zerumbone as a promising adjunctive neuroprotective agent against ferroptosis cell death in the context of cerebral stroke. This study lays the groundwork for subsequent in-depth investigations to fully elucidate its therapeutic potential in ischemic stroke treatment.

Histone Demethylase PHF8 Confers Protection against Oxidative Stress and Cardiomyocyte Apoptosis in Heart Failure by Upregulating FOXA2

Oxidative stress and cardiomyocyte apoptosis are hallmarks of heart failure (HF) development. Plant homeodomain finger protein 8 (PHF8) is a histone demethylase downregulated in failing human hearts. Nevertheless, the potential role of PHF8 in HF remains unclear. Therefore, this study aimed to explore the biological action and molecular mechanism of PHF8 in HF.A rat model of left anterior descending coronary artery (LAD) ligation-induced HF and a cardiomyocyte model of oxygen-glucose deprivation/reperfusion (OGD/R) were developed after gain- or loss-of-function experiments in rats and cardiomyocytes, respectively. Heart function indexes, such as left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular ejection fraction, and left ventricular fractional shortening, were detected. Changes in myocardial tissues were examined by pathological staining. Cardiomyocyte apoptosis and oxidative stress markers, such as malondialdehyde, reactive oxygen species, superoxide dismutase, and catalase, were examined. The relationship between PHF8 and forkhead box A2 (FOXA2) was analyzed by luciferase and chromatin immunoprecipitation-quantitative polymerase chain reaction assays.PHF8 was downregulated in LAD-ligated rats and OGD/R-exposed cardiomyocytes. Following PHF8 upregulation, pathological changes in myocardial tissues and heart dysfunction were improved in LAD-ligated rats. Importantly, cardiomyocyte apoptosis and oxidative stress were diminished in vivo and in vitro upon PHF8 upregulation. Mechanistically, PHF8 increased FOXA2 expression in a histone demethylase-dependent manner. FOXA2 silencing abrogated the protective effect of PHF8 upregulation on cardiomyocytes against OGD/R-induced apoptosis and oxidative stress.PHF8 exerts protective functions against cardiomyocyte apoptosis, oxidative stress, and heart dysfunction in HF, in correlation with FOXA2 upregulation. These results suggest that the PHF8/FOXA2 axis may be a promising therapeutic target to prevent HF.

The Study of the Protection Mechanism of Calycosin-7-O-β-d-Glucoside Against Oxygen–Glucose Deprivation/Reperfusion in HT22 Cells Based on Non-Targeted Metabolomics and Network Analysis

The cell non-targeted metabolomics technique was used to investigate the potential mechanism of Caly-cosin-7-O-β-d-glucoside (CAG) against cell oxygen–glucose deprivation/reperfusion (OGD/R). The OGD/R-injured HT22 cell model was constructed. The cells were divided into control, OGD/R, Edaravone (EDA), CAG-L, CAG-M, and CAG-H groups. The protective effect of CAG on OGD/R-injured nerve cells and its potential mechanism was investigated by detecting ROS levels, apoptosis rate, glutamic acid (Glu), γ-aminobutyric acid (GABA), nitric oxide (NO), and combining with cell non-targeted metabolomics. The results showed that after OGD/R, ROS levels, apoptosis rate, Glu and NO concentrations were significantly increased, while the concentrations of GABA were decreased considerably, which improved in a dose-dependent manner after CAG intervention. Cell non-targeted metabolomics results showed that CAG can dramatically improve the metabolomic characteristics of OGD/R-injured HT22 cells. Through bioinformatics analysis and molecular docking, it was found that purine metabolism may be an important pathway for CAG to treat OGD/R injury, and key proteins screened may be important targets for improving OGD/R injury. Therefore, CAG may protect OGD/R-injured HT22 cells by inhibiting apoptosis and oxidative stress, improving energy supply and the metabolomic characteristics of OGD/R-injured HT22 cells by regulating purine metabolism.

Trimetazidine: Activating AMPK Signal to Ameliorate Coronary Microcirculation Dysfunction after Myocardial Infarction.

Myocardial ischemia-reperfusion (I/R) injury and coronary microcirculation dysfunction (CMD) are observed in patients with myocardial infarction after vascular recanalization. The antianginal drug trimetazidine has been demonstrated to exert a protective effect in myocardial ischemia-reperfusion injury. This study aimed to investigate the role of trimetazidine in endothelial cell dysfunction caused by myocardial I/R injury and thus improve coronary microcirculation. The myocardial I/R mouse model was established, and trimetazidine was administered for 7 days before myocardial I/R model establishment. Echocardiography, 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H&E) staining, and thioflavin S staining were applied to assess myocardial injury and microvascular function. Additionally, the oxygen-glucose deprivation/reperfusion (OGD/R) model was developed in endothelial cells to simulate myocardial I/R injury in vitro. Griess reaction method, immunofluorescence, and western blotting (WB) were employed to detect the expressions of nitric oxide (NO), platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial (VE)-cadherin, zonula occludens protein 1 (ZO-1), occludin, vascular endothelial growth factor (VEGF) and adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling-related proteins in endothelial cells and mouse cardiomyocytes. AMPK pathway inhibitor compound C was used for further mechanism validation. Our research demonstrated that trimetazidine can alleviate myocardial pathological injury and cardiac function injury during myocardial I/R. Trimetazidine was observed to improve microvascular reflux phenomenon and microvascular function and barrier injury in myocardial I/R and OGD/R models. Additionally, the expressions of AMPK signal-related proteins were found to be inhibited in myocardial I/R and OGD/R models, which were then activated in mice administered trimetazidine. However, the effects of trimetazidine on endothelial cell function and barrier damage were attenuated after co-treatment with compound C and trimetazidine. Trimetazidine ameliorated myocardial I/R-induced CMD by activating AMPK signaling.

Naotaifang formula regulates Drp1-induced remodeling of mitochondrial dynamics following cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury (CIRI) has emerged as a hindrance for rehabilitation of ischemic stroke patients. Naotaifang (NTF) exhibits beneficial efficacy in alleviating inflammation and ferroptosis in vitro during CIRI. While the potential role of NTF in regulating mitochondrial dynamics in CIRI are not elucidated. This study aimed to explore the mechanism of NTF against CIRI by regulating the dynamin-related protein 1 (Drp1)-dependent mitochondrial fission/fusion. Modeling middle cerebral artery occlusion/reperfusion (MCAO/R) in vivo to evaluate the effects of NTF on the MCAO/R-damaged neurons and the structure, dynamics and function of mitochondria. An oxygen-glucose deprivation/reperfusion (OGD/R) cell model was established to evaluate the role of NTF in OGD/R-damaged cells. Function of Drp1 in CIRI and the neuroprotection of NTF through the mitochondrial fission/fusion pathway were investigated in vivo and in vitro. The results revealed that in vivo, NTF alleviated neuron injury in a dose-dependent manner, down-regulated Drp1 and fission protein 1 (Fis1) levels, upregulated optic atrophy 1 (Opa1), mitofusin 1/2 (Mfn1 and Mfn2), facilitated mitochondrial fusion and inhibited mitochondrial fission to rescue cells from CIRI. In vitro, Drp1 overexpression inhibited mitochondrial fusion and activated mitochondrial fission, while silencing of Drp1 exhibited the opposite result. NTF rebalanced mitochondrial dynamic in the OGD/R cell model. NTF could alleviate neuron injury following CIRI by regulating the balance of mitochondrial fission and fusion. Targeting Drp1-dependent mitochondrial dynamics may represent a viable treatment strategy for addressing the issues of CIRI post ischemic stroke.

Calycosin‑7‑O‑β‑D‑glucoside downregulates mitophagy by mitigating mitochondrial fission to protect HT22 cells from oxygen‑glucose deprivation/reperfusion‑induced injury.

Calycosin‑7‑O‑β‑D‑glucoside (CG), a major active ingredient of Astragali Radix, exerts neuroprotective effects against cerebral ischemia; however, whether the effects of CG are associated with mitochondrial protection remains unclear. The present study explored the role of CG in improving mitochondrial function in a HT22 cell model of oxygen‑glucose deprivation/reperfusion (OGD/R). The Cell Counting Kit‑8 assay, flow cytometry, immunofluorescence and western blotting were performed to investigate the effects of CG on mitochondrial function. The results demonstrated that mitochondrial function was restored after treatment with CG, as indicated by reduced mitochondrial reactive oxygen species levels, increased mitochondrial membrane potential and improved mitochondrial morphology. Overactivated mitophagy was revealed to be inhibited by the regulation of proteins involved in fission [phosphorylated‑dynamin‑related protein 1 (Drp1) and Drp1] and mitophagy (LC3, p62 and translocase of outer mitochondrial membrane 20), and mitochondrial biogenesis was demonstrated to be enhanced by increased levels of sirtuin 1 (SIRT1) and peroxisome proliferator‑activated receptor γ coactivator‑1α (PGC‑1α). In addition, neuronal apoptosis was ameliorated by CG, as determined by a decreased rate of apoptosis, and levels of caspase‑3 and Bcl‑2/Bax. In conclusion, the present study demonstrated that CG may alleviate OGD/R‑induced injury by upregulating SIRT1 and PGC‑1α protein expression, and reducing excessive mitochondrial fission and overactivation of mitophagy.

TGF-β1-induced apoptosis in retinal endothelial cells is implicated in retinal vein occlusion.

Retinal vein occlusion (RVO) is a serious vascular condition that impairs vision due to retinal endothelial cell injury and apoptosis. This study aimed to identify key molecular pathways and therapeutic targets involved in RVO pathogenesis. Transcriptomic analysis of the retinal tissues from a mouse RVO model was performed to identify differentially expressed genes and co-expression modules associated with RVO. Protein-protein interaction network analysis pinpointed putative hub genes. In vitro experiments using human retinal microvascular endothelial cells (HRMECs) validated the involvement of identified genes/pathways in apoptosis induced by oxygen-glucose deprivation/reperfusion (OGD/R) and UV exposure. Gene expression was assessed by RT-qPCR, while protein levels and phosphorylation were measured by ELISA and Western blotting. Apoptosis was evaluated using flow cytometry, and reactive oxygen species (ROS) were quantified using a fluorescence-based assay. A total of 392 genes were identified as putatively involved in RVO-associated apoptosis, enriched in MAPK, TGF-β and other signaling pathways. Among top hub genes, TGF-β1 emerged as a central regulator whose expression and signaling (pSmad2/3) increased after OGD/R induction or UV exposure in HRMECs. TGF-β1-induced HRMEC apoptosis was mediated by p38/JNK activation. Similar effects were observed for OGD/R and UV triggering TGF-β1-dependent p38/JNK signaling and apoptosis. Pharmacological inhibition of TGF-β signaling attenuated the apoptotic and oxidative stress responses induced by OGD/R and UV exposure. This study elucidates TGF-β1 as a crucial mediator of retinal endothelial injury through p38/JNK-induced apoptosis, suggesting TGF-β1 pathway inhibition as a potential therapeutic strategy for RVO.

Protective effects of PIK3CG knockdown against OGD/R-induced neuronal damage via inhibition of autophagy through the AMPK/mTOR pathway

BackgroundIschemic stroke represents an urgent need for more efficacious therapies owing to modest effectiveness of current treatment. MethodsDownload data from stroke patients and collect blood samples from clinical patients to analyze phosphatidylinositol-3 kinase catalytic subunit γ (PIK3CG) expression. To establish a brain damage model, oxygen glucose deprivation/reperfusion (OGD/R) was applied to SH-SY5Y cells. Impact of PIK3CG on AMPK/mTOR autophagy pathway was verified treating cells with AMPK activator metformin. Proliferation and apoptosis were identified by CCK8 and flow cytometry. ResultsDifferential expression analysis and clinical testing show that PIK3CG is highly expressed in patients. Prolonged ODG/R exposure increased PIK3CG levels, supressed cell proliferation, and induced apoptosis. KEGG pathway analysis implicated PIK3CG in autophagy pathway. Knockdown of PIK3CG supressed OGD/R-induced reductions in cell proliferation and OGD/R-induced increases in apoptosis and expressions of Beclin 1 and LC3 II. Following OGD/R, AMPK phosphorylation was upregulated while mammalian target of rapamycin (mTOR) phosphorylation was downregulated, indicating AMPK/mTOR autophagy activation. Knockdown of PIK3CG opposed metformin-induced rises in Beclin 1, LC3 II and apoptosis along with decreases in proliferation. ConclusionPIK3CG knockdown protects neuronal cells by inhibiting AMPK/mTOR autophagy pathway and further inhibiting autophagy.

Transcranial focused ultrasound stimulation alleviates NLRP3-related neuroinflammation induced by ischemic stroke via regulation of the Nespas/miR-383-3p/SHP2 pathway

Transcranial focused ultrasound stimulation (tFUS) has emerged as a promising therapeutic strategy for mitigating brain injury in animal models. In this study, the effects and mechanisms of tFUS on ischemic stroke were explored in a transient middle cerebral artery occlusion (MCAO) rat model. Low-intensity tFUS was administered to the ischemic hemisphere 24 h post-MCAO for seven consecutive days. Neurological function was evaluated through neurobehavioral assessments following tFUS treatment. Western blotting, immunofluorescence staining, and quantitative real-time PCR were performed to examine the impact of tFUS on NLRP3-related neuroinflammation using brain tissues from MCAO rats and BV2 cells subjected to oxygen glucose deprivation/reperfusion (OGD/R). Additionally, RNA sequencing and cell transient transfection were employed to elucidate the underlying mechanisms. The findings revealed that tFUS improved neurobehavioral performance, reduced infarct size, and suppressed NLRP3 inflammasome activation seven days post-MCAO. Notably, Nespas expression was significantly elevated in tFUS-treated rats, whereas Nespas silencing exacerbated neurological deficits and enhanced NLRP3 activation. Moreover, Nespas positively regulated src homology 2 domain-containing tyrosine phosphatase-2 (SHP2), and SHP2 inhibition significantly amplified NLRP3 activation. Mechanistic in vitro studies further demonstrated that Nespas attenuated microglial NLRP3 activation via the Nespas/miR-383-3p/SHP2 pathway. These results suggest that the neuroprotective effects of tFUS are likely mediated through the upregulation of Nespas and suppression of NLRP3 via the Nespas/miR-383-3p/SHP2 axis, offering new insights into the molecular mechanisms supporting tFUS as a potential therapeutic approach for stroke-induced brain injury.

BMSCs-derived small extracellular vesicles antagonize cerebral endothelial Caveolin-1 driven autophagic degradation of tight-junction proteins to protect blood-brain barrier post-stroke.

Bone marrow mesenchymal stem cells (BMSCs) -derived extracellular vesicles (EVs), especially small EVs (sEVs), were vastly reported to enable multiple restorative effects on ischemic stroke, yet the protective mechanism of blood-brain barrier (BBB) has not been fully illustrated. In the present study, we investigated the therapeutic effects and mechanism of BMSCs-derived sEVs on BBB injury after ischemic stroke. In-vivo, administering sEVs to transient middle cerebral artery occlusion (tMCAo) mice mitigated the brain infarct volume, BBB permeability and neural apoptosis, and improved the cerebral blood flow perfusion and neurological function. Simultaneously, cerebral vascular endothelial overexpressed Caveolin-1 (Cav-1) together with its strong co-localization with autophagosome protein LC3B were suppressed, and ZO-1 and Occludin expressions were enhanced, whose results were consistent with those of oxygen-glucose-deprivation/reperfusion (OGD/R)-insulted brain endothelial cells (BECs) in vitro. Furthermore, by employing Cav-1 siRNA and pcDNA3.1 transfection, Co-immunoprecipitation, cycloheximide assay, and molecular docking, it proved that brain endothelial Cav-1 was an essential upstream of autophagy activation, contributing to tight-junction proteins delegation via the autophagy-lysosomal pathway. Altogether, our study demonstrates the novel mechanism of Cav-1-dependent tight-junction proteins autophagic disruption on BBB integrity after ischemic stroke, and BMSC-sEVs treatment can reverse such hazard cascades.

CLEC7A Knockdown Alleviates Ischemic Stroke by Inhibiting Pyroptosis and Microglia Activation.

Ischemic stroke (IS) is the leading cause of mortality worldwide. Herein, we aimed to identify novel biomarkers and explore the role of C-type lectin domain family 7 member A (CLEC7A) in IS. Differentially expressed genes (DEGs) were screened using the GSE106680, GSE97537, and GSE61616 datasets, and hub genes were identified through construction of protein-protein interaction networks. An IS model was established by middle cerebral artery occlusion and reperfusion (MCAO/R). Neural function was assessed using triphenyl tetrazolium chloride, hematoxylin-eosin, and terminal deoxynucleotidyl transferase-mediated nick-end labeling. A cell counting kit was used to detect cell viability following oxygen-glucose deprivation/reperfusion (OGD/R). Inflammatory factors were detected using enzyme-linked immunosorbent assay. The mRNA and protein expression levels were detected using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. Fc fragment of Immunoglobulin G (IgG) receptor IIIa (FCGR3A), Fc fragment of Immunoglobulin E (IgE) receptor Ig (FCER1G), Complement component 5a receptor 1 (C5AR1), CLEC7A, Plasminogen activator, urokinase (PLAU), and C-C motif chemokine ligand 6 (CCL6) were identified as important hub genes, from which CLEC7A was selected as the primary subject of this study. The activation of microglia and pyroptosis were observed in MCAO/R model with increased levels of interleukin (IL)-1β, IL-18, tumor necrosis factor-α, and lactate dehydrogenase. CLEC7A knockdown was found to promote cell viability in BV2 cells and inhibiting pyroptosis in HT22 cells. CLEC7A knockdown in microglia also decreased infarct volume and neurological deficit scores, and alleviated injury and neuronal apoptosis in IS rats. CLEC7A knockdown inhibited pyroptosis and microglial activation in the MCAO/R model. A pyroptosis activator reversed the effect of CLEC7A knockdown on the viability of OGD/R-treated HT22 cells. CLEC7A is a promising biomarker of IS. CLEC7A knockdown alleviates IS by inhibiting pyroptosis and microglial activation.

NOD-like receptor X1 promotes autophagy and inactivates NLR family pyrin domain containing 3 inflammasome signaling by binding autophagy-related gene 5 to alleviate cerebral ischemia/reperfusion-induced neuronal injury.

Ischemic strokes pose serious risks to human health. We aimed to elucidate the function of NOD-like receptor X1 (NLRX1) in a rat middle cerebral artery occlusion (MCAO)-induced cerebral ischemia/reperfusion injury (CIRI) model and in an oxygen-glucose deprivation/reperfusion (OGD/R)-treated human microglial cell line (HMC3) model. Following NLRX1 upregulation, infarct volumes were measured with 2,3,5-triphenyltetrazolium chloride staining and pathological examination was conducted with hematoxylin-eosin staining. Results suggested that levels of NLRX1 were decreased in brain tissue of MCAO rats and in OGD/R-stimulated HMC3 cells. NOD-like receptor X1 overexpression mitigated the neuronal damage, reduced tumor necrosis factor-α and interleukin-6 expression, alleviated microglial activation, and induced autophagy in vivo and in vitro. Additionally, a coimmunoprecipitation assay indicated that NLRX1 bound to autophagy-related gene 5 (ATG5) to elevate ATG5 expression in HMC3 cells. Further, the elevated NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD, and cleaved caspase 1 expression in MCAO rats and HMC3 cells with OGD/R induction was reduced after NLRX1 upregulation. Importantly, ATG5 depletion abrogated the effects of NLRX1 elevation on NLRP3 inflammasome signaling. These results indicate that NLRX1 promotes autophagy and inactivates NLRP3 inflammasome signaling by binding ATG5 in experimental cerebral ischemia. These data may help the development of novel therapeutic strategies for ischemic stroke.

AEBP1 Silencing Protects Against Cerebral Ischemia/Reperfusion Injury by Regulating Neuron Ferroptosis and Microglia M2 Polarization Through PRKCA-PI3K-Akt Axis.

Cerebral ischemia/reperfusion injury is one of the main causes of neuronal damage. Neuron ferroptosis and microglia polarization are considered as critical processes during cerebral ischemia/reperfusion. Adipocyte enhancer-binding protein 1 (AEBP1) usually acts as a transcriptional repressor which is involved in various diseases. However, it is still remains unknown whether AEBP1 could have important roles in regulating the neuron ferroptosis and microglia polarization in cerebral ischemia/reperfusion injury. The oxygen-glucose deprivation and reperfusion (OGD/R)-treated cells and middle cerebral artery occlusion (MCAO)-treated mice were used as in vitro and in vivo models. The differentially expressed factors were analyzed according to GEO datasets. Relative mRNA and protein expression levels were detected by qRT-PCR and western blot analysis. Cell viability was measured by CCK-8 assay. ROS, GSH and iron contents were detected using specifical assay kits. CD26 and CD206 levels were measured by immunofluorescence assay. Inflammatory cytokines were detected by ELISA. The association between AEBP1 and PRKCA was assessed by luciferase reporter and ChIP analyses. The neuron damage in mice was analyzed by TTC staining and neurological deficit score. Transcription factor AEBP1 was increased in OGD/R-treated HT22 and BV2 cells. AEBP1 silencing attenuated OGD/R-induced HT22 cell ferroptosis through increasing cell viability, GSH and GPX4 levels, and decreasing ROS, iron and ACSL4 levels. AEBP1 knockdown promoted microglia M2 polarization by increasing CD206-positive cells and Arg-1 level, and reducing iNOS, TNF-α, IL-1β and IL-6 levels in BV2 cells. AEBP1 transcriptionally repressed PRKCA expression, and further regulated PI3K/Akt signaling activation. Inhibition of PRKCA or PI3K/Akt reversed the effects of AEBP1 silencing on neuron ferroptosis and microglia M2 polarization. AEBP1 downregulation attenuated neuronal damage by decreasing infarct size and deficit scores in MCAO-treated mice. AEBP1 silencing mitigated neuron ferroptosis and promoted microglia M2 polarization through increasing PRKCA and activating PI3K/Akt signaling, indicating the potentially protective action of AEBP1 knockdown in cerebral ischemia/reperfusion injury.

Unveiling Smyd-2's Role in Cytoplasmic Nrf-2 Sequestration and Ferroptosis Induction in Hippocampal Neurons After Cerebral Ischemia/Reperfusion.

SET and MYND Domain-Containing 2 (Smyd-2), a specific protein lysine methyltransferase (PKMT), influences both histones and non-histones. Its role in cerebral ischemia/reperfusion (CIR), particularly in ferroptosis-a regulated form of cell death driven by lipid peroxidation-remains poorly understood. This study identifies the expression of Smyd-2 in the brain and investigates its relationship with neuronal programmed cell death (PCD). We specifically investigated how Smyd-2 regulates ferroptosis in CIR through its interaction with the Nuclear Factor Erythroid-2-related Factor-2 (Nrf-2)/Kelch-like ECH-associated protein (Keap-1) pathway. Smyd-2 knockout protects HT-22 cells from Erastin-induced ferroptosis but not TNF-α + Smac-mimetic-induced apoptosis/necroptosis. This neuroprotective effect of Smyd-2 knockout in HT-22 cells after Oxygen-Glucose Deprivation/Reperfusion (OGD/R) was reversed by Erastin. Smyd-2 knockout in HT-22 cells shows neuroprotection primarily via the Nuclear Factor Erythroid-2-related Factor-2 (Nrf-2)/Kelch-like ECH-associated protein (Keap-1) pathway, despite the concurrent upregulation of Smyd-2 and Nrf-2 observed in both the middle cerebral artery occlusion (MCAO) and OGD/R models. Interestingly, vivo experiments demonstrated that Smyd-2 knockout significantly reduced ferroptosis and lipid peroxidation in hippocampal neurons following CIR. Moreover, the Nrf-2 inhibitor ML-385 abolished the neuroprotective effects of Smyd-2 knockout, confirming the pivotal role of Nrf-2 in ferroptosis regulation. Cycloheximide (CHX) fails to reduce Nrf-2 expression in Smyd-2 knockout HT-22 cells. Smyd-2 knockout suppresses Nrf-2 lysine methylation, thereby promoting the Nrf-2/Keap-1 pathway without affecting the PKC-δ/Nrf-2 pathway. Conversely, Smyd-2 overexpression disrupts Nrf-2 nuclear translocation, exacerbating ferroptosis and oxidative stress, highlighting its dual regulatory role. This study underscores Smyd-2's potential for ischemic stroke treatment by disrupting the Smyd-2/Nrf-2-driven antioxidant capacity, leading to hippocampal neuronal ferroptosis. By clarifying the intricate interplay between ferroptosis and oxidative stress via the Nrf-2/Keap-1 pathway, our findings provide new insights into the molecular mechanisms of CIR and identify Smyd-2 as a promising therapeutic target.

Algae-based flexible localized oxygen control around Cells: An approach leading to more Biomimetic microphysiological systems

Oxygen levels are crucial in the cellular microenvironment, affecting cellular function. Controlling pericellular oxygen is essential for understanding cell-oxygen interactions, disease mechanisms, and developing therapies. In this study, an algae strain was engineered as a biological oxygen-control material, integrated with a light-controlled microphysiological system to regulate pericellular oxygen. This technique could provide different oxygen levels and varying profiles for different locations within one microphysiological system. Additionally, the pericellular oxygen dynamics were faster than those achieved by other methods. By utilizing this algae-integrated microfluidic chip, mesenchymal stem cells differentiated into adipocytes with improved phenotypic characteristics, including increased numbers and sizes of lipid droplets, under 21-day stage-specific oxygen concentrations. Furthermore, this technique effectively mitigated the need to vent significant amounts of oxygen or nitrogen into the laboratory during long-term or virus-infested experiments, thereby enhancing both safety and practicality. The algae-integrated microfluidic chip also served as a platform for constructing a stroke-related vascular-on-chip model, enabling the evaluation of the protective effects of oxygen therapy on endothelial injury induced by oxygen-glucose deprivation/reperfusion (OGD/R). The results indicated that waving hyperoxic dynamics were most effective in safeguarding vascular tissue against OGD/R compared to other hyperoxic dynamics. This finding can guide the clinical oxygen inhalation mode during stroke management. All the aforementioned results demonstrated that the algae-light microphysiological system offered a technical platform capable of precise, dynamic pericellular oxygen control across the disciplines of biology and pharmaceutics. Moreover, The cost-effective cultivation of algae on a large scale made this method suitable for wider use.

Taxifolin attenuates hepatic ischemia-reperfusion injury by enhancing PINK1/Parkin-mediated mitophagy

BackgroundHepatic ischemia-reperfusion (I/R) injury stands as a recurring clinical challenge in liver transplantation, leading to mitochondrial dysfunction and cellular imbalance. Mitochondria, crucial for hepatocyte metabolism, are significantly damaged during hepatic I/R and the extent of mitochondrial damage correlates with hepatocyte injury. PINK1/Parkin-mediated mitophagy, is a specialized form of cellular autophagy, that maintains mitochondrial quality by identifying and removing damaged mitochondria, thereby restoring cellular homeostasis. Taxifolin (TAX), a natural flavonoid, possesses antioxidant, anti-inflammatory and anticancer properties. This study aimed at investigating the effects of TAX on hepatic I/R and the underlying mechanisms. MethodsC57BL/6 mice were pretreated with TAX or vehicle control, followed by 60 min of 70% hepatic ischemia. After 6 h of reperfusion, the mice were euthanized. In vitro, TAX-pretreated primary hepatocytes were subjected to oxygen glucose deprivation/reperfusion (OGD/R). ResultsHepatic I/R caused mitochondrial damage and apoptosis in hepatocytes, but TAX pretreatment mitigated these effects by normalizing mitochondrial membrane potential and inhibiting reducing apoptotic protein expression. TAX exerted its protective effects by enhancing mitophagy via the PINK1/Parkin pathway. Moreover, silencing the PINK1 gene in primary hepatocytes reversed the beneficial effects of TAX. ConclusionThe results of the study demonstrate that promoting mitophagy through the PINK1/Parkin pathway restores mitochondrial function and protects the liver from I/R, suggesting that it may have therapeutic potential for the treatment of hepatic I/R.

Gnetupendin A protects against ischemic stroke through activating the PI3K/AKT/mTOR-dependent autophagy pathway

BackgroundAutophagy has been recently emerged as a prominent factor in the pathogenesis of ischemic stroke (IS) and is increasingly being considered as a potential therapeutic target for IS. Gnetum parvifolium has been identified as a potential therapeutic agent for inflammatory diseases such as rheumatism and traumatic injuries. However, the pharmacological effects of Gnetupindin A (GA), a stilbene compound isolated from Gnetum parvifolium, have not been fully elucidated until now. ObjectiveHere we identified the therapeutic potential of GA for IS, deeply exploring the possible mechanisms related to its regulation of autophagy. MethodsThe mouse model of middle cerebral artery occlusion-reperfusion (MCAO/R) and the oxygen-glucose deprivation reperfusion (OGD/R)-exposed cells served as models to study the protection of GA against IS. The adeno-associated virus (AAV) encoding shAtg5, in conjunction with autophagy inhibitor 3-Methyladenine (3-MA) were utilized to explore the role of GA in regulating autophagy following IS. Molecular docking, CETSA, and DARTS were used to identify the specific therapeutic target of GA. PI3K inhibitor LY294002 was employed to test the participation of PI3K in GA-mediated autophagy and neuroprotective effects following IS. ResultsOur findings revealed that treatment with GA significantly alleviated the brain infract volume, edema, improved neurological deficits and attenuated apoptosis. Mechanistically, we found that GA promoted autophagic flow both in vivo and in vitro after IS. Notably, neural-targeted knockdown of Atg5 abolished the neuroprotective effects mediated by GA. Inhibition of autophagy using 3-MA blocked the attenuation on apoptosis induced by GA. Moreover, molecular docking, CETSA, and DARTS analysis demonstrated that GA specifically targeted PI3K and further inhibited the activation of PI3K/AKT/mTOR signaling pathway. LY294002, which inhibits PI3K, reversed GA-induced autophagy and neuroprotective effects on OGD/R-treated cells. ConclusionWe demonstrated, for the first time, that GA protects against IS through promoting the PI3K/AKT/mTOR-dependent autophagy pathway. Our findings provide a novel mechanistic insight into the anti-IS effect of GA in regulating autophagy.

BAK ameliorated cerebral infarction/ischemia–reperfusion injury by activating AMPK/Nrf2 to inhibit TXNIP/NLRP3/caspase-1 axis

BackgroundCerebral ischemia/reperfusion (I/R) injury is a serious vascular disease with extremely high mortality and disability rate. Bakuchiol (BAK) was found in leaves and seeds of Psoralea corylifolia Linn and has been shown to decrease inflammation and reduce oxidative stress, while the mechanism of BAK in ameliorating cerebral I/R injury remains unclear. MethodsMiddle cerebral artery occlusion reperfusion (MACO/R) was used to establish mouse model. The protective effect of BAK in MCAO/R mices was detected by performing neurological deficit testing, TTC staining, and H&E staining. Oxygen/glucose deprivation and reperfusion (OGD/R) was used to stimulate SH-SY5Y cells in vitro. Protein expression was detected by western blotting, gene expression was detected by quantitative real-time polymerase chain reaction and apoptosis was detected by immunofluorescence. ResultsOur study indicated that BAK protected ischemia–reperfusion injury in MACO/R mice, and upregulated superoxide dismutase (SOD) and the catalase (CAT) enzyme activity. BAK also inhibited the expression of TNF-α, IL-1β, IL-6, and IL-18 and suppressed apoptosis and pyroptosis both in MACO/R mice and in OGD/R SH-SY5Y cells. Further results showed that BAK could suppress TXNIP, ASC, NLRP3, and caspase-1 mRNA levels to reverse assembly of inflammasome. And BAK could also upregulate the expression of phosphorylated AMP-activated protein kinase (AMPK) and nuclear factor erythroid 2-related factor (Nrf2). In addition, Nrf2 inhibitor ML385 reversed the BAK induced reduction of TXNIP, ASC, NLRP3, and the AMPK inhibitor also abolished BAK’ the effect on the regulation of Nrf2, TXNIP, ASC, NLRP3, caspase-1, and pro-inflammatory cytokines. In conclusion, BAK, found in leaves and seeds of Psoralea corylifolia Linn, could ameliorated cerebral I/R injury through activating AMPK/Nrf2 to inhibit NLRP3 inflammasome, which might present new therapeutic strategy for cerebral I/R injury.

E3 Ubiquitin Ligase Ring Finger Protein 2 Alleviates Cerebral Ischemia-Reperfusion Injury by Stabilizing Mesencephalic Astrocyte-Derived Neurotrophic Factor Through Monoubiquitination.

Cerebral ischemic stroke (IS) is one of the leading causes of morbidity and mortality globally. However, the mechanisms underlying IS injury remain poorly understood. Ring finger protein 2 (RNF2), the member of the polycomb family (PcG), has been implicated in diverse biological and pathological conditions. However, whether RNF2 plays a role in IS progression is not clarified. This study aims to investigate the potential effects of RNF2 on IS. The effects of RNF2 were studied in human postmortem IS brains, a rat model of IS, tunicamycin (TM)-induced mouse neuroblastoma neuro2a (N2a) cells, and oxygen-glucose deprivation/reperfusion (OGD/R)-induced SH-SY5Y cells. Here, we demonstrated that RNF2 was markedly upregulated both in human postmortem IS brains and ischemic rat brains and RNF2 overexpression alleviated brain injury induced by middle cerebral artery occlusion by reducing neuron apoptosis. Mechanistically, we found that RNF2 is an E3 ubiquitin ligase for the mesencephalic astrocyte-derived neurotrophic factor (MANF), which confers protection against brain ischemia. RNF2 interacted with MANF and promoted the monoubiquitination of MANF, consequently facilitating its stability and nuclear localization. Collectively, RNF2 is identified as a critical inhibitor of IS injury by stabilizing MANF through monoubiquitination, suggesting that RNF2 is a potential therapeutic target for IS.

Polygala tenuifolia root extract attenuates ischemic stroke by inhibiting HMGB1 trigger neuroinflammation

Polygala tenuifolia Willd., a famous traditional Chinese medicine, has been widely applied to treat central nervous system diseases. In this study, P. tenuifolia root extract exhibited a moderate anti-ischemic effect on in-vitro oxygen-glucose deprivation/reperfusion (OGD/R) model. In transient middle cerebral artery occlusion (tMCAO) rats, P. tenuifolia root extract significantly attenuated brain infarction and neurological deficits in a dose-dependent manner. Compared with the sham group, the release of damage-associated molecular patterns (DAMPs)-HMGB1 in the ischemic brain was significantly higher, which was inhibited by P. tenuifolia root extract. To further explore such neuroprotective effects whether associated with aseptic inflammation, HMGB1-activated BV2 microglial cells model was established. The extract of P. tenuifolia was found to inhibit the downstream inflammatory response driven by HMGB1, with an IC50 value of 49.46 μg/mL. In addition, the extract was also found to be able to directly interact with HMGB1 in the surface plasmon resonance (SPR) experiment. Phytochemical studies showed that the extract of P. tenuifolia root contains a large number of terpenoids, oligosaccharides and phenolic compounds, which likely contribute to the above observed biological activities. Our results not only provide some data support for the clinical application of P. tenuifolia against cerebral ischemia, but also clarify the potential target of P tenuifolia's anti-inflammatory properties.