Oxygen-glucose Deprivation/reoxygenation Research Articles

Activation of GPR30 Ameliorates Cerebral Ischemia-Reperfusion Injury by Suppressing Ferroptosis Through Nrf2/GPX4 Signaling Pathway.

The newly identified estrogen receptor, G protein-coupled receptor 30 (GPR30), is prevalent in the brain and has been shown to provide significant neuroprotection. Recent studies have linked ferroptosis, a newly characterized form of programmed cell death, closely with cerebral ischemia-reperfusion injury (CIRI), highlighting it as a major contributing factor. Consequently, our research aimed to explore the potential of GPR30 targeting in controlling neuronal ferroptosis and lessening CIRI impacts. Results indicated that GPR30 activation not only improved neurological outcomes and decreased infarct size in a mouse model but also lessened iron accumulation and malondialdehyde formation post-middle cerebral artery occlusion (MCAO). This protective effect extended to increased levels of Nrf2 and GPX4 proteins. Similar protective results were replicated in PC12 cells subjected to Oxygen Glucose Deprivation and Reoxygenation (OGD/R) using the GPR30-specific agonist G1. Importantly, inhibition of Nrf2 with ML385 curtailed the neuroprotective effects of GPR30 activation, suggesting that GPR30 mitigates CIRI primarily through inhibition of neuronal ferroptosis via upregulation of Nrf2 and GPX4.

Palladium–reduced graphene oxide nanocomposites enhance neurite outgrowth and protect neurons from Ishemic stroke

Currently, the construction of novel biomimetic reduced graphene oxide (RGO)-based nanocomposites to induce neurite sprouting and repair the injured neurons represents a promising strategy in promoting neuronal development or treatment of cerebral anoxia or ischemia. Here, we present an effective method for constructing palladium-reduced graphene oxide (Pd-RGO) nanocomposites by covalently bonding Pd onto RGO surfaces to enhance neurite sprouting of cultured neurons. As described, the Pd-RGO nanocomposites exhibit the required physicochemical features for better biocompatibility without impacting cell viability. Primary neurons cultured on Pd-RGO nanocomposites had significantly increased number and length of neuronal processes, including both axons and dendrites, compared with the control. Western blotting showed that Pd-RGO nanocomposites improved the expression levels of growth associate protein-43 (GAP-43), as well as β-III tubulin, Tau-1, microtubule-associated protein-2 (MAP2), four proteins that are involved in regulating neurite sprouting and outgrowth. Importantly, Pd-RGO significantly promoted neurite length and complexity under oxygen-glucose deprivation/re-oxygenation (OGD/R) conditions, an in vitro cellular model of ischemic brain damage, that closely relates to neuronal GAP-43 expression. Furthermore, using the middle cerebral artery occlusion (MCAO) model in rats, we found Pd-RGO effectively reduced the infarct area, decreased neuronal apoptosis in the brain, and improved the rats’ behavioral outcomes after MCAO. Together, these results indicate the great potential of Pd-RGO nanocomposites as a novel excellent biomimetic material for neural interfacing that shed light on its applications in brain injuries.

Safflower Yellow Injection Alleviates Myocardial Ischemia/Reperfusion Injury by Reducing Oxidative and Endoplasmic Reticulum Stress.

Safflower yellow is an extract of the famous Chinese medicine Carthamus tinctorious L, and safflower yellow injection (SYI) is widely used clinically to treat angina pectoris. However, there are few studies on the anti-myocardial ischemia/reperfusion (I/R) injury effect of SYI, and its mechanisms are unclear. In the present study, we aimed to investigate the protective effect of SYI on myocardial I/R injury and explore its underlying mechanisms. Male Sprague Dawley rats were randomly divided into a control group, sham group, model group, and SYI group (20 mg/kg, femoral vein injection 1 h before modeling). The left anterior descending coronary artery was ligated to establish a myocardial I/R model. H9c2 cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) after incubation with 80 μg/mL SYI for 24 h. In vivo, TsTC, HE, and TUNEL staining were performed to evaluate myocardial injury and apoptosis. A kit was used to detect superoxide dismutase (SOD) and malondialdehyde (MDA) to assess oxidative stress. In vitro, flow cytometry was used to detect the reactive oxygen species (ROS) content and apoptosis rate. Protein levels were determined via Western blotting. Pretreatment with SYI significantly reduced infarct size and pathological damage in rat hearts and suppressed cardiomyocyte apoptosis in vivo and in vitro. In addition, SYI inhibited oxidative stress by increasing SOD activity and decreasing MDA content and ROS production. Myocardial I/R and OGD/R activate endoplasmic reticulum (ER) stress, as evidenced by increased expression of activating transcription factor 6 (ATF6), glucose-regulated protein 78 (GRP78), cysteinyl aspartate-specific proteinase caspase-12, and C/EBP-homologous protein (CHOP), which were all inhibited by SYI. SYI ameliorated myocardial I/R injury by attenuating apoptosis, oxidative damage, and ER stress, which revealed new mechanistic insights into its application.

Ginsenoside Rg1 ameliorates cerebral ischemia-reperfusion injury by regulating Pink1/ Parkin-mediated mitochondrial autophagy and inhibiting microglia NLRP3 activation

ObjectiveThis study aimed to further elucidate the mechanism of ginsenoside Rg1 in the treatment of cerebral ischemia-reperfusion. MethodsIn this study, we observed the apoptosis of RM cells (microglia) after oxygen-glucose deprivation/reoxygenation (OGD/R) modeling before and after Rg1 administration, changes in mitochondrial membrane potential, changes in the content of Reactive oxygen species (ROS) and inflammatory vesicles NLR Family Pyrin Domain Containing 3 (NLRP3), and the expression levels of autophagy-related proteins, inflammatory factors, and apoptosis proteins. We further examined the pathomorphological changes in brain tissue, neuronal damage, changes in mitochondrial morphology and mitochondrial structure, and the autophagy-related proteins, inflammatory factors, and apoptosis proteins expression levels in CI/RI rats before and after administration of Rg1 in vivo experiments. ResultsIn vitro experiments showed that Rg1 induced mitochondrial autophagy, decreased mitochondrial membrane potential, and reduced ROS content thereby inhibiting NLRP3 activation, decreasing secretion of inflammatory factors and RM cell apoptosis by regulating the PTEN induced putative kinase 1(Pink1) /Parkin signaling pathway. In vivo experiments showed that Rg1 induced mitochondrial autophagy, inhibited NLRP3 activation, improved inflammatory response, and reduced apoptosis by regulating the Pink1/Parkin signaling pathway, and Rg1 significantly reduced the area of cerebral infarcts, improved the pathological state of brain tissue, and attenuated the neuronal damage, thus improving cerebral ischemia/reperfusion injury in rats. ConclusionOur results suggest that ginsenoside Rg1 can ameliorate cerebral ischemia-reperfusion injury by modulating Pink1/ Parkin-mediated mitochondrial autophagy in microglia and inhibiting microglial NLRP3 activation.

An engineered cellular carrier delivers miR-138–5p to enhance mitophagy and protect hypoxic-injured neurons via the DNMT3A/Rhebl1 axis

Mitophagy influences the progression and prognosis of ischemic stroke (IS). However, whether DNA methylation in the brain is associated with altered mitophagy in hypoxia-injured neurons remains unclear. Here, miR-138–5p was found to be highly expressed in exosomes secreted by astrocytes stimulated with oxygen and glucose deprivation/re-oxygenation (OGD/R), which could influence the recovery of OGD/R-injured neurons through autophagy. Mechanistically, miR-138–5p promotes the stable expression of Ras homolog enriched in brain like 1(Rhebl1) through DNA-methyltransferase-3a (DNMT3A), thereby enhancing ubiquitin-dependent mitophagy to maintain mitochondrial homeostasis. Furthermore, we employed glycosylation engineering and bioorthogonal click reactions to load mirna onto the surface of microglia and deliver them to injured region utilising the inflammatory chemotactic properties of microglia to achieve drug-targeted delivery to the central nervous system (CNS). Our findings demonstrate miR-138–5p improves mitochondrial function in neurons through the miR-138–5p/DNMT3A/Rhebl1 axis. Additionally, our engineered cell vector-targeted delivery system could be promising for treating IS. Statement of significanceIn this study, we demonstrated that miR-138–5p in exosomes secreted by astrocytes under hypoxia plays a critical role in the treatment of hypoxia-injured neurons. And we find a new target of miR-138–5p, DNMT3A, which affects neuronal mitophagy and thus exerts a protective effect by regulating the methylation of Rbebl1. Furthermore, we have developed a carrier delivery system by combining miR-138–5p with the cell membrane of microglia and utilized the inflammatory chemotactic properties of microglia to deliver this system to the brain via intravenous injection. This groundbreaking study not only provides a novel therapeutic approach for ischemia-reperfusion treatment but also establishes a solid theoretical foundation for further research on targeted drug delivery for central nervous system diseases with promising clinical applications.

Pharmacologic activation of activating transcription factor 6 contributes to neuronal survival after spinal cord injury in mice.

The impact of primary and secondary injuries of spinal cord injury (SCI) results in the demise of numerous neurons, and there is still no efficacious pharmacological intervention for it. Recently, studies have shown that endoplasmic reticulum stress (ERS) plays a pivotal role in recovery of neurological function after spinal cord injury. As a process to cope with intracellular accumulation of misfolded and unfolded proteins which triggers ERS, the unfolded protein response (UPR) plays an important role in maintaining protein homeostasis. And, a recently disclosed small molecule AA147, which selectively activates activating transcription factor 6 (ATF6), has shown promising pharmacological effects in several disease models. Thus, it seems feasible to protect the neurons after spinal cord injury by modulating UPR. In this study, primary neurons were isolated from E17-19 C57BL/6J mouse embryos and we observed that AA147 effectively promoted the survival of neurons and alleviated neuronal apoptosis after oxygen-glucose deprivation/reoxygenation (OGD/R) invitro. This was evident through a decrease in the proportion of PI-positive and TUNEL-positive cells, an increase in BCL-2 expression, and a decrease in the expression of BAX and C-caspase3. In in-vivo experiments, these findings were corroborated by TUNEL staining and immunohistochemistry. It was also found that AA147 enhanced three arms of the unfolded protein response with reduced CHOP expression. Besides, AA147 mitigated the accumulation of ROS in neurons probably by upregulating catalase expression. Furthermore, spinal cord injury models of C57BL/6J mice were established and behavioral experiments revealed that AA147 facilitated the recovery of motor function following SCI. Thus, pharmacologic activation of ATF6 represents a promise therapeutic approach to ameliorate the prognosis of SCI.

β -estradiol alleviates Hypoxic-ischemic brain damage in neonatal rats through the GPER1 regulating AKT/NF-κB signal pathway

IntroductionResearch has established that estradiol (E2) offers neuroprotection against hypoxic-ischemic brain damage (HIBD) in neonatal rats, yet the underlying mechanisms are not fully understood. This study seeks to delineate whether E2's neuroprotective effects in neonatal HIBD are mediated through astrocytes by modulating the G Protein-Coupled Estrogen Receptor 1 (GPER1) receptor and the subsequent AKT Serine (AKT)/NF-κB signaling cascade.Material and methodsWe developed an in vivo HIBD model in neonatal rats and established primary cultures of astrocytes subjected to oxygen-glucose deprivation-reoxygenation (OGD-R) as an in vitro model. E2 and the GPER1 inhibitor (G15) were administered according to the experimental design. Protein expression levels of GPER1, phosphorylated AKT (p-AKT), NF-κB p65, and cleaved-caspase3 were examined using Western blot analysis. Apoptosis was assessed via the TUNEL assay, and the presence of TNF-α and IL-1β in the cell supernatant was quantified by ELISA. The localization of p-AKT and NF-κB p65 was determined through immunofluorescence.ResultsOur findings indicate that E2 treatment significantly reduced the volume of brain infarction and astrocyte apoptosis. E2 upregulated GPER1 and p-AKT expression while downregulating NF-κB p65 and cleaved-caspase3 levels in astrocytes and neonatal rats post-HIBD. Additionally, E2 diminished the secretion of TNF-α and IL-1β in the cell supernatant. The G15 inhibitor notably reversed the neuroprotective effects of E2 and the associated molecular changes.ConclusionsThese results suggest that E2 may exert neuroprotection in neonatal rats with HIBD by inhibiting astrocyte apoptosis and modulating the expression of GPER1, p-AKT, and NF-κB, thereby providing a potential therapeutic strategy for HIBD.

Exosomal miR-486 derived from bone marrow mesenchymal stem cells promotes angiogenesis following cerebral ischemic injury by regulating the PTEN/Akt pathway

Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) have been shown to promote angiogenesis after ischemic stroke, in which microRNAs (miRs) are believed to play an important role in exosome-mediated therapeutic effects, though the mechanism is still not clear. In this study, a series of molecular biological and cellular assays, both in vitro and in vivo, were performed to elucidate the role of exosomal miR-486 in angiogenesis following cerebral ischemic and its molecular mechanisms. Our results revealed that BMSC-Exos significantly improved neurological function and increased microvessel density in ischemic stroke rats. In vitro assays showed that BMSC-Exos promoted the proliferation, migration, and tube formation ability of oxygen–glucose deprivation/reoxygenation (OGD/R) injured rat brain microvascular endothelial cells (RBMECs). Importantly, BMSC-Exos increased the expression of miR-486 and phosphorylated protein kinase B (p-Akt) and down-regulated the protein level of phosphatase and tensin homolog (PTEN) in vivo and in vitro. Mechanistic studies demonstrated that transfection with miR-486 mimic enhanced RBMECs angiogenesis and increased p-Akt expression, while inhibited PTEN expression. On the other hand, the miR-486 inhibitor induced an opposite effect, which could be blocked by PTEN siRNA. It was thus concluded that exosomal miR-486 from BMSCs may enhance the functional recovery by promoting angiogenesis following cerebral ischemic injury, which might be related to its regulation of the PTEN/Akt pathway.

Overexpression of MiR-188-5p Downregulates IL6ST/STAT3/ NLRP3 Pathway to Ameliorate Neuron Injury in Oxygen-glucose Deprivation/Reoxygenation.

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. This study investigates the role of miR-188-5p in hippocampal neuron cell injury associated with Cerebral Ischemia-Reperfusion (CI/R). 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. 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. 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.

Mechanism of ligustilide in attenuating OGD/R-induced HT22 cell injury based on FtMt inhibition of ferroptosis

This study investigated the role and mechanism of ligustilide(LIG) in attenuating oxygen-glucose deprivation/reoxyge-nation(OGD/R)-induced damage to mouse hippocampal neuron cells(HT22) by inhibiting ferroptosis through mitochondrial ferritin(FtMt). An in vitro model of OGD/R-induced HT22 cell damage was established. HT22 cells were randomly divided into normal group, model group, LIG groups(5, 10, and 20 μmol·L~(-1)), and ferrostatin-1(Fer-1, 2 μmol·L~(-1)) group. Cell viability was mea-sured using the CCK-8 method, and lactate dehydrogenase(LDH) release was measured using an LDH assay kit. Cell morphology was observed under an inverted microscope, and mitochondrial ultrastructure was observed using transmission electron microscopy. Intracellular Fe~(2+) content was detected using a chemiluminescence method. To further investigate the mechanism of FtMt inhibition of ferroptosis, FtMt in HT22 cells was silenced and divided into normal group, model group, LIG group(20 μmol·L~(-1)), si-NC group, si-FtMt group, and si-FtMt+20 μmol·L~(-1) LIG group. Immunofluorescence and Western blot were used to detect FtMt expression. Chemiluminescence was used to measure the content of NADPH/NADP~+, GSH, MDA, and ATP in HT22 cells. The mtROS fluorescence intensity was observed by laser confocal microscopy, and intracellular Fe~(2+) content was measured by flow cytometry. The expression of ferroptosis-related proteins Ferrtin, GPX4, and ACSL4 was detected by Western blot. The results showed that compared with the model group, LIG significantly increased the survival rate of HT22 cells, improved the morphology of damaged HT22 cells and mitochondrial ultrastructure, decreased intracellular Fe~(2+) content, and reduced the expression of the pro-ferroptosis protein ACSL4 while increasing the expression of anti-ferroptosis proteins Ferrtin and GPX4. After silencing FtMt, LIG promoted FtMt expression. Compared with the si-FtMt group, LIG significantly increased the content of NADPH/NADP~+ and GSH, reduced mtROS fluorescence intensity and MDA content, increased ATP activity, decreased intracellular Fe~(2+) content, inhibited the expression of pro-ferroptosis protein ACSL4, and increased the expression of anti-ferroptosis proteins Ferrtin and GPX4. In summary, LIG improved mitochondrial function by upregula-ting FtMt expression to inhibit ferroptosis, thereby alleviating OGD/R-induced damage to HT22 cells.

LINC00894 Regulates Cerebral Ischemia/Reperfusion Injury by Stabilizing EIF5 and Facilitating ATF4-Mediated Induction of FGF21 and ACOD1 Expression

The non-coding RNA LINC00894 modulates tumor proliferation and drug resistance. However, its role in brain is still unclear. Using RNA-pull down combined with mass spectrometry and RNA binding protein immunoprecipitation, EIF5 was identified to interact with LINC00894. Furthermore, LINC00894 knockdown decreased EIF5 protein expression, whereas LINC00894 overexpression increased EIF5 protein expression in SH-SY5Y and BE(2)-M17 (M17) neuroblastoma cells. Additionally, LINC00894 affected the ubiquitination modification of EIF5. Adeno-associated virus (AAV) mediated LINC00894 overexpression in the brain inhibited the expression of activated Caspase-3, while increased EIF5 protein level in rats and mice subjected to transient middle cerebral artery occlusion reperfusion (MCAO/R). Meanwhile, LINC00894 knockdown increased the number of apoptotic cells and expression of activated Caspase-3, and its overexpression decreased them in the oxygen–glucose deprivation and reoxygenation (OGD/R) in vitro models. Further, LINC00894 was revealed to regulated ATF4 protein expression in condition of OGD/R and normoxia. LINC00894 knockdown also decreased the expression of glutamate-cysteine ligase catalytic subunit (GCLC) and ATF4, downregulated glutathione (GSH), and the ratio of GSH to oxidized GSH (GSH: GSSG) in vitro. By using RNA-seq combined with qRT-PCR and immunoblot, we identified that fibroblast growth factor 21 (FGF21) and aconitate decarboxylase 1 (ACOD1), as the ATF4 target genes were regulated by LINC00894 in the MCAO/R model. Finally, we revealed that ATF4 transcriptionally regulated FGF21 and ACOD1 expression; ectopic overexpression of FGF21 or ACOD1 in LINC00894 knockdown cells decreased activated Caspase-3 expression in the OGD/R model. Our results demonstrated that LINC00894 regulated cerebral ischemia injury by stabilizing EIF5 and facilitating EIF5-ATF4-dependent induction of FGF21 and ACOD1.

Discovery of New Phenyltetrazolium Derivatives as Ferroptosis Inhibitors for Treating Ischemic Stroke: An Example Development from Free Radical Scavengers.

Ferroptosis is a promising therapeutic target for injury-related diseases, yet diversity in ferroptosis inhibitors remains limited. In this study, initial structure optimization led us to focus on the bond dissociation enthalpy (BDE) of the N-H bond and the residency time of radical scavengers in a phospholipid bilayer, which may play an important role in ferroptosis inhibition potency. This led to the discovery of compound D1, exhibiting potent ferroptosis inhibition, high radical scavenging, and moderate membrane permeability. D1 demonstrated significant neuroprotection in an oxygen glucose deprivation/reoxygenation (OGD/R) model and reduced infarct volume in an in vivo stroke model upon intravenous treatment. Further screening based on this strategy identified NecroX-7 and Eriodictyol-7-O-glucoside as novel ferroptosis inhibitors with highly polar structural characteristics. This approach bridges the gap between free radical scavengers and ferroptosis inhibitors, providing a foundation for research and insights into novel ferroptosis inhibitor development.

Inhibition of Hsp90 K284 Acetylation Aalleviates Cardiac Injury After Ischemia-Reperfusion Injury.

Our objective was to determine the role of acetyl-Hsp90 and its relationship with the NF-κB p65 signaling pathway in CVDs. We investigated the effect of acetyl-Hsp90 on cardiac inflammation and apoptosis after ischemia-reperfusion injury (I/RI). The results showed that the induction of acetyl-Hsp90 occurred in the heart during I/R and in primary cardiomyocytes during oxygen-glucose deprivation/reoxygenation (OGD/R). Moreover, the nonacetylated mutant of Hsp90 (Hsp90-K284R), through the regulation of ATPase activities within its N-terminal domain (NTD), indirectly or directly increases its interaction with NF-κB p65. This led to a reduction in the activation of the NF-κB p65 pathway, thereby attenuating inflammation, apoptosis, and fibrosis, ultimately leading to an improvement in cardiac function. Furthermore, we demonstrated that recombinant human interleukin-37 (rIL-37) exerts a similar cardioprotective effect by reducing acetylation at K284 of Hsp90 after inhibiting the expression of KAT2A.

Modulating the RPS27A/PSMD12/NF-κB pathway to control immune response in mouse brain ischemia-reperfusion injury

BackgroundInvestigating immune cell infiltration in the brain post-ischemia-reperfusion (I/R) injury is crucial for understanding and managing the resultant inflammatory responses. This study aims to unravel the role of the RPS27A-mediated PSMD12/NF-κB axis in controlling immune cell infiltration in the context of cerebral I/R injury.MethodsTo identify genes associated with cerebral I/R injury, high-throughput sequencing was employed. The potential downstream genes were further analyzed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein-Protein Interaction (PPI) analyses. For experimental models, primary microglia and neurons were extracted from the cortical tissues of mouse brains. An in vitro cerebral I/R injury model was established in microglia using the oxygen-glucose deprivation/reoxygenation (OGD/R) technique. In vivo models involved inducing cerebral I/R injury in mice through the middle cerebral artery occlusion (MCAO) method. These models were used to assess neurological function, immune cell infiltration, and inflammatory factor release.ResultsThe study identified RPS27A as a key player in cerebral I/R injury, with PSMD12 likely acting as its downstream regulator. Silencing RPS27A in OGD/R-induced microglia decreased the release of inflammatory factors and reduced neuron apoptosis. Additionally, RPS27A silencing in cerebral cortex tissues mediated the PSMD12/NF-κB axis, resulting in decreased inflammatory factor release, reduced neutrophil infiltration, and improved cerebral injury outcomes in I/R-injured mice.ConclusionRPS27A regulates the expression of the PSMD12/NF-κB signaling axis, leading to the induction of inflammatory factors in microglial cells, promoting immune cell infiltration in brain tissue, and exacerbating brain damage in I/R mice. This study introduces novel insights and theoretical foundations for the treatment of nerve damage caused by I/R, suggesting that targeting the RPS27A and downstream PSMD12/NF-κB signaling axis for drug development could represent a new direction in I/R therapy.

ARL13B promotes cell cycle through the sonic hedgehog signaling pathway to alleviate nerve damage during cerebral ischemia/reperfusion in rats

Cerebral ischemia/reperfusion (CIRI) is a leading cause of death worldwide. A small GTPase known as ADP-ribosylation factor-like protein 13B (ARL13B) is essential in several illnesses. The role of ARL13B in CIRI remains unknown, though. A middle cerebral artery occlusion/reperfusion (MCAO/R) in rats as well as an oxygen-glucose deprivation/reoxygenation (OGD/R) models in PC12 cells were constructed. The neuroprotective effects of ARL13B against MCAO/R were evaluated using neurological scores, TTC staining, rotarod testing, H&E staining, and Nissl staining. To detect the expression of proteins associated with the SHH pathway and apoptosis, western blotting and immunofluorescence were employed. Apoptosis was detected using TUNEL assays and flow cytometry. There was increased expression of ARL13B in cerebral ischemia/reperfusion models. However, ARL13B knockdown aggravated CIRI nerve injury by inhibiting the sonic hedgehog (SHH) pathway. In addition, the use of SHH pathway agonist (SAG) can increased ARL13B expression, reverse the effects of ARL13B knockdown exacerbating CIRI nerve injury. ARL13B alleviated cerebral infarction and pathological injury and played a protective role against MCAO/R. Furthermore, ARL13B significantly increased the expression of SHH pathway-related proteins and the anti-apoptotic protein BCL-2, while decreased the expression of pro-apoptotic protein BAX, thus reducing apoptosis. The results from the OGD/R model in PC12 cells were consistent with those obtained in vivo. Surprisingly, we demonstrated that ARL13B regulates the cell cycle to protect against CIRI nerve injury. Our findings indicate that ARL13B protects against CIRI by reducing apoptosis through SHH-dependent pathway activation, and suggest that ARL13B plays a crucial role in CIRI pathogenesis.

The influence of ferroptosis on the in vitro OGD/R model in rat microglia

ABSTRACT Objective We aimed to explore the influence of ferroptosis on an oxygen-glucose deprivation/reoxygenation (OGD/R) model in primary rat microglia. Methods Primary microglia were extracted from rats and cultured in vitro. The cells were subjected to a hypoxic environment for 6 h in a glucose-free medium, and then re-oxygenated for 24 h in DMEM/F12. Rat microglia were pretreated with the ferroptosis activator erastin and the ferroptosis inhibitor ferrostatin 1 for 24 h, followed by detection of cell cycle progression and apoptosis by flow cytometry. Intracellular total iron levels were measured. In addition, the relative levels of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were determined using enzyme-linked immunosorbent assay. The protein levels of 15-lox2, GPX4, SLC7A11, ACSL4, and TFR1 were examined by western blotting. Results Compared with rat microglia subjected to OGD/R, pretreatment with erastin did not influence cell apoptosis but significantly enhanced total iron levels, MDA, and ROS levels, whereas it reduced SOD levels. Moreover, it upregulated ACSL4, TFR1, and 15-lox2 and downregulated GPX4 and SLC7A11. Pretreatment with ferrostatin 1 significantly inhibited cell apoptosis and cell cycle arrest in the G0/G1 phase. It significantly reduced total iron levels, MDA, and ROS levels and enhanced SOD levels, which also downregulated ACSL4, TFR1, and 15-lox2, and upregulated GPX4 and SLC7A11. Conclusion Our study showed that inhibition of ferroptosis is favorable against potential OGD/R-induced damage in rat microglia.

Buyang Huanwu Decoction suppresses ischemic stroke by suppressing glycolysis and cell apoptosis in rat brain microvascular endothelial cells

BackgroundBuyang Huanwu Decoction (BHD) is widely used in Chinese clinical practice for the treatment and prevention of ischemic cerebral vascular diseases. This study was designed to investigate the effects of BHD on ischemic stroke (IS) and its underlying mechanism. MethodsThe middle cerebral artery occlusion (MCAO) rat model and oxygen-glucose deprivation and reoxygenation (OGD/R) rat brain microvascular endothelial cell (RBMVEC) models were established. Brain infarction size and neurological score were calculated following MCAO surgery. Evans blue was used to measure blood brain barrier (BBB) permeability. Cell counting kit-8 (CCK-8) and TUNEL assays were performed to evaluate the cell viability and apoptosis of RBMVECs. Dual-luciferase reporter assay was used to analyze the transcriptional activities of apoptosis-related genes. ResultsResults showed that higher infarction volume, neurological scores, and BBB permeability in the MCAO group rats were reduced after BHD treatment. Drug serum (DS) treatment had no impact on the normal RBMVECs’ cell viability and cell apoptosis. Besides, DS treatment decreased the lactate production, glucose uptake, and extracellular acidification rate in normal and OGD/R-induced RBMVECs. DS treatment downregulated the protein levels of pan-lysine lactylation (kla), histone H3 lysine 18 lactylation (H3K18la), and the transcriptional of apoptotic protease activating factor-1 (Apaf-1) in OGD/R-treated RBMVECs. In addition, Apaf-1 overexpression decreased cell viability and increased apoptosis and glycolysis activity of OGD/R-treated RBMVECs. ConclusionIn summary, BHD inhibited glycolysis and apoptosis via suppressing the pan-kla and H3K18la protein levels and the Apaf-1 transcriptional activity, thus restraining the progression of IS.

Runx1 promotes neuronal injury in ischemic stroke through mediating miR-203-3p/Pde4d axis

ABSTRACT Background It has been reported that Runx1 engaged in IS progression, but the detailed mechanism of Runx1 in IS is still unclear. Methods Mice and HT22 cells were subjected to the process of middle cerebral artery occlusion and reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R), respectively. Infract volume was tested using TTC staining. The levels of inflammatory cytokines were investigated using ELISA assay. Cell viability was examined utilizing MTS. Apoptosis rate was evaluated using flow cytometry and TUNEL. The productions of SOD and MDA were monitored by means of commercial kits. The correlations among Runx1, miR-203-3p and Pde4d were ascertained using dual luciferase reporter gene, ChIP and RNA-RNA pull-down assays. Results Runx1 and Pde4d were abnormally elevated, while miR-203-3p was notably declined in MCAO/R mice and OGD/R-induced HT22 cells. OGD/R treatment suppressed cell viability and facilitated cell apoptosis, inflammation and oxidative stress, which were compromised by Runx1 knockdown or miR-203-3p upregulation. Runx1 bound to miR-203-3p promoter, thus decreasing miR-203-3p expression. MiR-203-3p inhibited Pde4d expression via targeting Pde4d mRNA. Runx1 deficiency-induced protection effects on OGD/R-treated HT22 cells were offset by miR-203-3p downregulation. Conclusion Runx1 aggravated neuronal injury caused by IS through mediating miR-203-3p/Pde4d axis.

Neuroprotective and vasoprotective effects of herb pair of Zhiqiao-Danggui in ischemic stroke uncovered by LC-MS/MS-based metabolomics approach.

Ischemic stroke is the most important cause of disability and death worldwide, but current treatments remain limited. Traditional Chinese medicine (TCM) including the herb pair of Zhiqiao-Danggui (ZD) offers a multifaceted treatment approach through promoting blood circulation, yet its specific anti-ischemic mechanism remains unclear. This study used the photochemically induced thrombosis (PIT) mouse model and the oxygen glucose deprivation/reoxygenation (OGD/R) cell model to explore the therapeutic effect of ZD on ischemic stroke. Mice were treated with high and low doses of ZD extract or positive control. Behavior was assessed using the grid test. The brain tissue was then subjected to infarct volume assessment, histopathology, oxidative stress marker detection, LC/MS metabolomic analysis and qRT-PCR validation. The therapeutic effect of ZD-medicated serum on OGD/R model was tested on cells. Experimental results show that ZD can improve motor function, reduce infarct size, neuronal damage and apoptosis as well as alleviate oxidative stress in mice. ZD-medicated serum promotes endothelial cell proliferation, improves cell survival against OGD/R-induced injury, reduces oxidative damage and protects mitochondrial function. Metabolomics reveals ZD regulation of metabolites in energy metabolism, amino acid metabolism, TCA cycle, and angiogenesis signaling pathways. qRT-PCR results also showed that ZD could attenuate abnormal conduction of angiogenic signals and enhance vessel stability. This study confirmed the neuroprotective and vasoprotective effects of ZD, highlighted its potential in treating ischemic stroke, and provided a scientific basis for the traditional use of ZD.

Shionone relieves oxygen-glucose deprivation/reoxygenation induced SH-SY5Y cells injury by inhibiting the p38 MAPK/NF-κB pathway

BackgroundCerebral ischemia-reperfusion injury (I/R) can affect patient outcomes and can even be life-threatening. This study aimed to explore the role of Shionone in cerebral I/R and reveal its mechanism of action through the cerebral I/R in vitro model.MethodsSH-SY5Y cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to induce cerebral I/R in vitro model. SH-SY5Y cells were treated with different concentrations of Shionone. Cell counting kit-8 and flow cytometry assays were used to detect cell viability and apoptosis levels. The levels of superoxide dismutase, catalase, and malondialdehyde were determined using their corresponding kits to examine the level of oxidative stress. The inflammation response was detected by IL-6, IL-1β, and TNF-α levels, using enzyme-linked-immunosorbent-assay. RT-qPCR was performed to measure the mRNA levels of p38 and NF-κB. Western blotting was used to quantify the apoptosis-related proteins and p38MAPK/NF-κB signaling pathway proteins.ResultsShionone exhibited no toxic effects on SH-SY5Y cells. Shionone inhibited OGD/R-induced cell apoptosis, improved the inflammatory response caused by OGD/R, and reduced the level of oxidative stress in cells. Western blot assay results showed that Shionone alleviated OGD/R-induced injury by inhibiting the activity of the p38 MAPK/NF-κB signaling pathway. The p38/MAPK agonist P79350 reversed the beneficial effects of Shionone.ConclusionShionone alleviates cerebral I/R and may thus be a novel therapeutic strategy for treating cerebral I/R.