Oxygen-glucose Deprivation/reoxygenation Research Articles

STIM2 Suppression Blocks Glial Activation to Alleviate Brain Ischemia Reperfusion Injury via Inhibition of Inflammation and Pyroptosis.

Cerebral ischemia/reperfusion injury (CIRI) involves various pathogenic mechanisms, including cytotoxicity, apoptosis, inflammation, and pyroptosis. Stromal interactive molecule 2 (STIM2) is implicated in cerebral ischemia. Consequently, this study investigates the biological functions of STIM2 and its related mechanisms in CIRI progression. Middle cerebral artery occlusion/reperfusion (MCAO/R) mouse models and oxygen-glucose deprivation/reoxygenation (OGD/R) cellular models were established. STIM2 level was upregulated in experimental CIRI models, as shown by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting and immunofluorescence staining. Brain infarction and edema were attenuated by STIM2 knockdown, as 2,3,5-triphenyltetrazolium chloride (TTC) staining and brain water content evaluation revealed. STIM2 knockdown relieved neuronal apoptosis, microglia activation, inflammation and pyroptosis in MCAO/R mice, as detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, enzyme-linked immunosorbent assay (ELISA) and western blotting. Results of flow cytometry, ELISA, western blotting and cell counting kit-8 (CCK-8) assays also showed that STIM2 knockdown inhibited inflammation, apoptosis and pyroptosis in OGD/R-treated BV2 cells. Moreover, STIM2 knockdown inhibited apoptosis and pyroptosis in PC12 cells incubated with conditioned medium collected from OGD/R-exposed BV2 cells. Mechanistically, lncRNA Malat1 (metastasis associated lung adenocarcinoma transcript 1) positively regulated STIM2 expression by sponging miR-30d-5p. Their binding relationship was confirmed by luciferase reporter assays. Finally, lncRNA Malat1 elevation or miR-30d-5p knockdown abolished the sh-STIM2-induced inhibition in cell damage. In conclusion, STIM2 knockdown in microglia alleviates CIRI by inhibiting microglial activation, inflammation, apoptosis, and pyroptosis.

HET0016 inhibits neuronal pyroptosis in the immature brain post-TBI via the p38 MAPK signaling pathway

Traumatic brain injury (TBI) is a serious health threat worldwide, especially for the younger demographic. Our previous study demonstrated that HET0016 (a specific inhibitor of 20-hydroxyeicosatetraenoic acid synthesis) can decrease the lesion volume in the immature brain post-TBI; however, its mechanism of action and its association with pyroptosis post-TBI are unclear. In this study, we established a controlled cortical impact (CCI) injury rat model (postnatal day 9–10) and observed that increased expression of indicators for pyroptosis, including NLR family pyrin domain containing 3 (NLRP3), caspase-1 and gasdermin D (GSDMD) proteins and interleukin (IL)-18/IL-1β mRNA during the acute phase of TBI, especially on post-injury day (PID) 1. Additionally, we found that caspase-1 was primarily expressed in the neurons and microglia. HET0016 (1 mg/kg/d, ip, 3 consecutive days since TBI) reduced the lesion volume; neuronal death; expression of NLRP3, caspase-1, and GSDMD; and expression of IL-18/IL-1β mRNA. Bioinformatics analysis suggested involvement of mitogen-activated protein kinase (MAPK) signaling pathway in the HET0016-mediated neuroprotective role against TBI in the immature brain. Western blot analysis revealed reduced expression of p-p38 MAPK and nuclear factor-kappa B (NF-κB) p65 in the neurons and microglia upon HET0016 treatment in TBI rats. In cultured primary cortical neurons subjected to oxygen-glucose deprivation/re-oxygenation (OGD) + (lipopolysaccharide) LPS, HET0016-induced the reduction of p-p38 MAPK, NLRP3, cleaved-caspase-1, GSDMD, IL-18, and IL-1β was reversed by co-treatment with p38 MAPK activator as well as NLRP3 agonist. Therefore, we conclude that pyroptosis is involved in neuronal death in the immature brains post-TBI and that HET0016 administration can alleviate neuronal pyroptosis possibly via inhibiting the phosphorylation of p38 MAPK.

LncRNA Gm44206 Promotes Microglial Pyroptosis Through NLRP3/Caspase-1/GSDMD Axis and Aggravate Cerebral Ischemia-Reperfusion Injury.

Inhibition of the inflammatory response triggered by microglial pyroptosis inflammatory activation may be one of the effective ways to alleviate cerebral ischemia-reperfusion injury, the specific mechanism of which remains unclear. In this study, BV-2 microglia with or without oxygen-glucose deprivation/reoxygenation (OGD/R) or long noncoding RNA (lncRNA) Gm44206 knockdown were used as cell models to conduct an in vitro study. Detection of lactate dehydrogenase release and pyroptosis-related protein levels was performed using a corresponding kit and western blotting, respectively. Proliferation of microglia was evaluated by CCK8 assay. Enzyme-linked immunosorbent assay was applied for measuring levels of proinflammatory cytokines. This study verified the involvement of microglial pyroptosis as well as upregulation of NLRP3, Caspase-1, GSDMD, and Apoptosis-associated Speck-like protein containing a C-terminal caspase-recruitment domain (ASC) in cerebral ischemia-reperfusion injury. Moreover, knockdown of lncRNA Gm44206 could alleviate OGD/R-induced microglial pyroptosis and cell proliferation inhibition through the NLRP3/Caspase-1/GSDMD pathway, thus decreasing the release of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-18, and tumor necrosis factor-alpha. In conclusion, this study established a correlation between microglial pyroptosis and cerebral ischemia-reperfusion injury and identified lncRNA Gm44206 as a potential regulator of NLRP3/Caspase-1/GSDMD axis-mediated microglial pyroptosis, which could be considered a promising therapeutic target.

Calycosin attenuates the inflammatory damage of microglia induced by oxygen and glucose deprivation through the HMGB1/TLR4/NF-κB signaling pathway.

Stroke seriously threatens human life and health worldwide, but only very few effective stroke medicines are currently available. Our previous studies have indicated that the phytoestrogen calycosin exerts neuroprotective effects in cerebral ischemia and reperfusion injury rats. Therefore, the objective of this study is to further explore the protective effect of calycosin on inflammatory injury in microglia after oxygen-glucose deprivation/reoxygenation (OGD/R) and to clarify whether its protective effect is related to the HMGB1/TLR4/NF-κB signaling pathway. Here, the OGD/R model of rodent microglia is established in vitro to simulate cerebral ischemia-reperfusion injury. Through the CCK-8 test, ELISA, qRT-PCR, and western blot analysis, we find that the activity of microglia is decreased, the expressions of HMGB1 and TLR4 and the phosphorylation of NF-κB (p-NF-κB) are increased, and the releases of the inflammatory factors IL-6, IL-1β, and TNF-α are increased after OGD/R. Pretreatment with calycosin could ameliorate these states, increase cell viability, reduce HMGB1, TLR4 and p-NF-κB expression, and reduce inflammatory cytokine production. In addition, the effect of calycosin is similar to that of TAK-242 (an inhibitor of TLR4), and the effect of the combined treatment is better than that of the single treatment. The results indicate that calycosin protects microglia from OGD/R injury and reduces the inflammatory response. Calycosin might alleviate cerebral ischemia-reperfusion injury by inhibiting the HMGB1/TLR4/NF-κB pathway.

Ursolic Acid Ameliorated Neuronal Damage by Restoring Microglia-Activated MMP/TIMP Imbalance in vitro.

The oxygen and glucose deprivation-reoxygenation (OGDR) model is widely used to evaluate ischemic stroke and cerebral ischemia-reperfusion (I/R) injury in vitro. Excessively activated microglia produce pro-inflammatory mediators such as matrix metalloproteinases [MMPs] and their specific inhibitors, tissue inhibitors of metalloproteinases [TIMPs], causing neuronal damage. Ursolic acid (UA) acts as a neuroprotective agent in the rat middle cerebral artery occlusion/reperfusion (MCAO/R) model keeping the MMP/TIMP balance with underlying mechanisms unclear. Our study used OGDR model to determine whether and how UA reduces neuronal damage by reversing MMP/TIMP imbalance caused by microglia in I/R injury in vitro. SH-SY5Y cells were first cultured with 95% N2 and 5% CO2 and then cultivated regularly for OGDR model. Cell viability was tested for a proper UA dose. We established a co-culture system with SH-SY5Y cells and microglia-conditioned medium (MCM) stimulated by lipopolysaccharide (LPS) and interferon-gamma (IFNγ). MMP9 and TIMP1 levels were measured with ELISA assay to confirm the UA effect. We added recombinant MMP9 (rMMP9) and TIMP1 neutralizing antibody (anti-TIMP1) for reconfirmation. Transmission electron microscopy was used to observe cell morphology, and flow cytometry and Annexin V-FITC and PI labeling for apoptotic conditions. We further measured the calcium fluorescence intensity in SH-SY5Y cells. The MCM significantly reduced cell viability of SH-SY5Y cells after OGDR (p<0.01), which was restored by UA (0.25 µM) (p<0.05), whereas lactate dehydrogenase activity, intraneuronal Ca2+ concentration, and apoptosis-related indexes were showed significant improvement after UA treatment (p<0.01). UA corrected the MMP/TIMP imbalance by decreasing MMP9 expression and increasing TIMP1 expression in the co-culture system (p<0.01) and the effects of UA on SH-SY5Y cells were mitigated by the administration of rMMP9 and anti-TIMP1 (p<0.01). We demonstrated that UA inhibited microglia-induced neuronal cell death in an OGDR model of ischemic reperfusion injury by stabilizing the MMP9/TIMP1 imbalance.

EHMT2 affects microglia polarization and aggravates neuronal damage and inflammatory response via regulating HMOX1.

This research was designed to ascertain the function of euchromatic histone lysine methyltransferase 2 (EHMT2) in ischemic stroke-induced neuronal damage and inflammatory response and its regulatory mechanism. Mouse microglia (BV-2 cells) were induced by oxygen glucose deprivation/reoxygenation (OGD/R) to establish a cellular model, and then co-cultured with HT22 hippocampal neurons. After that, HT22 cell viability and apoptosis were evaluated, followed by the measurement of apoptosis-related factors (B-cell lymphoma-2, Bcl-2 associated X, and cleaved-Caspase 3). Meanwhile, the expression of inducible nitric oxide synthase (M1 microglia polarization marker) and arginase 1 (M2 microglia polarization marker) in BV-2 cells was detected, as well as the levels of inflammatory factors (tumor necrosis factor-α, interleukin [IL]-6, IL-10, IL-1β, and IL-4). Additionally, the expression of EHMT2 and heme oxygenase 1 (HMOX1) in BV-2 cells was assessed by quantitative reverse transcription polymerase chain reaction and western blot, and the binding between EHMT2 and HMOX1 was predicted and verified. OGD/R treatment led to decreased cell viability and increased cell apoptosis in HT22 cells, and aggravated inflammatory response in BV-2 cells. In OGD/R-induced BV-2 cells, EHMT2 and HMOX1 were increasingly expressed, and knockdown of EHMT2 or HMOX1 in BV-2 cells could inhibit neuronal damage and inflammatory response. Moreover, EHMT2 promoted HMOX1 transcription level by histone methylation. Collected evidence showed that down-regulation of EHMT2 relieved neuronal damage and inflammatory response by inhibiting HMOX1 expression.

NDUFC2 deficiency exacerbates endothelial mesenchymal transformation during ischemia-reperfusion via NLRP3.

Ischemic stroke is the main type of cerebrovascular disease. Emergency thrombectomy combined with medication therapy is currently the primary treatment for stroke. Inflammation and oxidative stress induced by ischemia-reperfusion cause secondary damage to blood vessels, especially endothelial mesenchymal transformation (EndoMT). However, much is still unclear about the role of EndoMT in ischemia-reperfusion. In this study, an in vivo ischemia-reperfusion model was established by transient middle cerebral artery occlusion (tMCAO) in wild-type (WT) C57BL/6 mice and NLRP3 (NOD-like receptor thermal protein domain associated protein 3) knockout (KO) C57BL/6 mice. An in vitro ischemia-reperfusion model was established by oxygen glucose deprivation and reoxygenation (OGD/R) of human brain microvascular endothelial cells (HBMECs). α-SMA (alpha smooth muscle actin), CD31 (platelet endothelial cell adhesion molecule-1, PECAM-1/CD31), NDUFC2 (NADH: ubiquinone oxidoreductase subunit C2), and NLRP3 were used to evaluate EndoMT and inflammation. Real-time PCR measured superoxide dismutase 1 (SOD1) and catalase (CAT) mRNA expression to evaluate oxidative stress levels. NLRP3 was activated by ischemia-reperfusion injury and NLRP3 inactivation inhibited the EndoMT in tMCAO mice. Further experiments demonstrated that OGD/R treatment induced NLRP3 activation and EndoMT in HBMECs, which resulted in NDUFC2 deficiency. NDUFC2 overexpression suppressed NLRP3 activation and EndoMT in HBMECs induced by OGD/R. Moreover, NDUFC2 overexpression rescued SOD1 and CAT mRNA expression. These results demonstrated that NDUFC2 deficiency decreased the antioxidant levels, leading to NLRP3 activation and EndoMT during ischemia-reperfusion injury and suggesting that NDUFC2 is a potential drug target for the treatment of ischemic stroke.

Overexpression of FSP1 Ameliorates ferroptosis via PI3K/ AKT /GSK3β pathway in PC12 cells with Oxygen-Glucose Deprivation/Reoxygenation

After ischemia and reperfusion (I/R), nerve cell damage is a pathogenic process that involves numerous molecular processes. In the last ten years, one new classification of programmed cell death is ferroptosis. More recent research has demonstrated that ferroptosis has a role in a variety of neurological disorders, including stroke, cancer, and neurodegenerative illnesses. Ferroptosis suppressor protein 1 (FSP1) plays a significant role in inhibiting ferroptosis. The purpose of this work is to determine how overexpression of FSP1 affects the ferroptosis of PC12 cells under the condition of oxygen-glucose deprivation/reoxygenation (OGD/R). The expression of FSP1 was regulated by lentivirus transfection technology. Western blot and immunofluorescence were used to measure protein levels related to ferroptosis and the PI3K/AKT/GSK3β signal pathway. Determine cell viability using the appropriate kit. Mitochondrial structural morphology was checked by transmission electron microscopy in PC12 cells. Reactive oxygen species (ROS) and Malondialdehyde (MDA) were quantified using the relevant kits. OGD/R induced ferroptosis in PC12 cells, however, FSP1 overexpression reverses ferroptosis and promotes cell viability, lowering ROS and MDA content. The expression of FSP1 decreased in OGD/R0h and OGD/R6h and rebounded in OGD/R24h and OGD/R48h. During the processes of OGD/R-induced ferroptosis, FSP1 overexpression significantly stimulated PI3K/AKT/GSK3β pathway, but LY294002 weakens the protective effect of FSP1 overexpression. Our outcomes demonstrate that overexpression of FSP1 markedly enhances the ability to resist ferroptosis via the PI3K/AKT/GSK3β pathway. The above results may provide a new preliminary lead for the treatment of the cerebral ischemia-reperfusion injury.

Neuroprotective Effects of Activin A against Cerebral Ischemia/Reperfusion Injury in Mice by Enhancing Nrf2 Expression to Attenuate Neuronal Ferroptosis.

Activin A (Act A) is a member of the transforming growth factor-β (TGF-β) superfamily and can protect against ischemic cerebral injury. Ferroptosis, a newly discovered type of programmed cell death, contributes to the pathogenesis of cerebral ischemia-reperfusion injury (CIRI). However, little is known on whether Act A can modulate neuronal ferroptosis to protect against CIRI in a mouse model of middle cerebral artery occlusion (MCAO) and an HT22 cell model of oxygen-glucose deprivation/reoxygenation (OGD/R). The results indicated that Act A treatment relieved CIRI by improving neurological deficits and reducing the infarct volume in mice. MCAO stimulated iron accumulation and malondialdehyde formation and upregulated ACSL4 expression but downregulated GPX4 expression, a hallmark of ferroptosis in the brain of mice. Treatment with Act A significantly mitigated MCAO-triggered ferroptosis in the brain of mice. Furthermore, Act A treatment enhanced the MCAO-upregulated nuclear factor erythroid-2-related factor 2 (Nrf2) expression in the brains of mice. Similar results were observed in HT22 cells following OGD/R and pretreatment with Act A. The neuronal protective effect of Act A in HT22 cells was attenuated by treatment with ML385, an Nrf2 inhibitor. To conclude, Act A attenuated CIRI by enhancing Nrf2 expression and inhibiting neuronal ferroptosis.

LncRNA MALAT1 Promoted Neuronal Necroptosis in Cerebral Ischemia-reperfusion Mice by Stabilizing HSP90.

The objective of this research was to investigate the role of lncRNA MALAT1 and HSP90 in the regulation of neuronal necroptosis in mice with cerebral ischemia-reperfusion (CIR). We used male C57BL/6J mice to establish a middle cerebral artery occlusion (MCAO) model and conducted in vitro experiments using the HT-22 mouse hippocampal neuron cell line. The cellular localization of NeuN and MLKL, as well as the expression levels of neuronal necroptosis factors, MALAT1, and HSP90 were analyzed. Cell viability and necroptosis were assessed, and we also investigated the relationship between MALAT1 and HSP90. The results showed that MALAT1 expression increased after MCAO and oxygen-glucose deprivation/re-oxygenation (OGD/R) treatment in both cerebral tissues and cells compared with the control group. The levels of neuronal necroptosis factors and the co-localization of NeuN and MLKL were also increased in MCAO mice compared with the Sham group. MALAT1 was found to interact with HSP90, and inhibition of HSP90 expression led to decreased phosphorylation levels of neuronal necroptosis factors. Inhibition of MALAT1 expression resulted in decreased co-localization levels of NeuN and MLKL, decreased phosphorylation levels of neuronal necroptosis factors, and reduced necroptosis rate in cerebral tissues. Furthermore, inhibiting MALAT1 expression also led to a shorter half-life of HSP90, increased ubiquitination level, and decreased phosphorylation levels of neuronal necroptosis factors in cells. In conclusion, this study demonstrated that lncRNA MALAT1 promotes neuronal necroptosis in CIR mice by stabilizing HSP90.

Knockdown of Mmu-circ-0001380 Attenuates Myocardial Ischemia/Reperfusion Injury via Modulating miR-106b-5p/Phlpp2 Axis.

Myocardial ischemia/reperfusion (MI/R) injury induces myocardial damage and dysfunction. Increasing evidence has confirmed that circular RNAs (circRNAs) play crucial roles in regulating MI/R. Mmu-circ-0001380 has identified to be highly expressed in myocardium of MI/R mouse model. However, its biological function and molecular mechanism in MI/R injury are still unclear. Here, we demonstrated that knockdown of cric-0001380 attenuated myocardial injury of MI/R mice. In vitro, silence of circ-0001380 significantly enhanced viability, and inhibited apoptosis and oxidative stress in HL-1 cells under oxygen-glucose deprivation/reoxygenation (OGD/R). Mmu-miR-106b-5p interacted with circ-0001380, and suppressed the expression of pleckstrin homology domain and leucine rich repeat protein phosphatase 2 (Phlpp2). The miR-106b-5p/Phlpp2 axis mediated the effect of circ-0001380 on OGD/R-induced apoptosis through regulating the phosphorylation of p38, and further involved in regulating the viability and oxidative stress of HL-1 cells. In conclusion, circ-0001380 downregulation relieves MI/R injury via regulating the miR-106b-5p/Phlpp2 axis. The present study indicates that mmu-circ-0001380 exacerbates the myocardial ischemia/reperfusion injury through modulating the miR-106b-5p/Phlpp2 axis in vitro and in vivo.

L-Arginine-Loaded Gold Nanocages Ameliorate Myocardial Ischemia/Reperfusion Injury by Promoting Nitric Oxide Production and Maintaining Mitochondrial Function.

Cardiovascular disease is the leading cause of death worldwide. Reperfusion therapy is vital to patient survival after a heart attack but can cause myocardial ischemia/reperfusion injury (MI/RI). Nitric oxide (NO) can ameliorate MI/RI and is a key molecule for drug development. However, reactive oxygen species (ROS) can easily oxidize NO to peroxynitrite, which causes secondary cardiomyocyte damage. Herein, L-arginine-loaded selenium-coated gold nanocages (AAS) are designed, synthesized, and modified with PCM (WLSEAGPVVTVRALRGTGSW) to obtain AASP, which targets cardiomyocytes, exhibits increased cellular uptake, and improves photoacoustic imaging in vitro and in vivo. AASP significantly inhibits oxygen glucose deprivation/reoxygenation (OGD/R)-induced H9C2 cell cytotoxicity and apoptosis. Mechanistic investigation revealed that AASP improves mitochondrial membrane potential (MMP), restores ATP synthase activity, blocks ROS generation, and prevents NO oxidation, and NO blocks ROS release by regulating the closing of the mitochondrial permeability transition pore (mPTP). AASP administration in vivo improves myocardial function, inhibits myocardial apoptosis and fibrosis, and ultimately attenuates MI/RI in rats by maintaining mitochondrial function and regulating NO signaling. AASP shows good safety and biocompatibility in vivo. This findings confirm the rational design of AASP, which can provide effective treatment for MI/RI.

Dan-Deng-Tong-Nao softgel capsule promotes angiogenesis of cerebral microvasculature to protect cerebral ischemia reperfusion injury via activating HIF-1α-VEGFA-Notch1 signaling pathway

BackgroundA proprietary Chinese herbal product called Dan-Deng-Tong-Nao softgel capsule (DDTNC) is used to treat ischemic stroke. However, the preventive mechanisms of DDTNC against cerebral ischemia reperfusion injury (CIRI) haven not been characterized. ObjectiveTo explore the mechanisms of protective effects of DDTNC against CIRI from both internal and external levels. MethodsChemical characterization was performed using UPLC. The potential protective mechanisms of DDTNC against CIRI were predicted using network pharmacology. Model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established in rats. An model of brain microvascular endothelial cells (BMECs) induced by oxygen-glucose deprivation/reoxygenation (OGD/R) was also established. We evaluated neurological deficits, cerebral infarct volume, cortical neuron damage, and mitochondrial swelling in vivo. We evaluated the expression of VEGFR2, VEGFA, HIF-1α, CD31, and CD34 in ischemic cortex, and VEGF, bFGF, BDNF, angiostatin, and endostatin in serum of rats and in BMEC supernatants. We also evaluated cell viability, cytotoxicity, intracellular ROS, apoptosis, and migration ability in vitro. ResultsSeven components were detected in DDTNC. KEGG enrichment analysis showed that DDTNC may modulate angiogenesis via the HIF-1 signaling pathway. DDTNC treatment reduced neurological score and infarct volume, and improved cell morphology of damaged neurons. Transmission electron microscopy showed that DDTNC reduced mitochondria swelling in cortical neurons. Furthermore, DDTNC reduced intracellular ROS and inhibited apoptosis. DDTNC boosted the expression of CD31, CD34, VEGFR2, VEGFA and HIF-1α, highlighting its involvement in angiogenesis, according to immunofluorescence studies. Furthermore, DDTNC enhanced tube formation and migration of BMECs in vitro. ELISA and western blotting indicated that DDTNCCSF induced the expression of VEGF, BDNF and bFGF, reduced the level of angiostatin and endostatin, increased the protein expression of VEGFA, Notch1 and HIF-1α in vitro and in vivo. ConclusionsDDTNC promoted angiogenesis to protect brain tissue against MCAO/R, and exerted protective effects against OGD/R in BMECs via activating HIF-1α-VEGFA-NOTCH1 signal transduction pathway.

Sodium Tanshinone IIA Sulfonate Ameliorates Oxygen-glucose Deprivation/Reoxygenation-induced Neuronal Injury via Protection of Mitochondria and Promotion of Autophagy.

Sodium tanshinone IIA sulfonate (STS) has shown significant clinical therapeutic effects in cerebral ischemic stroke (CIS), but the molecular mechanisms of neuroprotection remain partially known. The purpose of this study was to explore whether STS plays a protective role in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury by regulating microglia autophagy and inflammatory activity. Co-cultured microglia and neurons were subjected to OGD/R injury, an in vitro model of ischemia/reperfusion (I/R) injury with or without STS treatment. Expression of protein phosphatase 2A (PP2A) and autophagy-associated proteins Beclin 1, autophagy related 5 (ATG5), and p62 in microglia was determined by Western blotting. Autophagic flux in microglia was observed with confocal laser scanning microscopy. Neuronal apoptosis was measured by flow cytometric and TUNEL assays. Neuronal mitochondrial function was determined via assessments of reactive oxygen species generation and mitochondrial membrane potential integrity. STS treatment markedly induced PP2A expression in microglia. Forced overexpression of PP2A increased levels of Beclin 1 and ATG5, decreased the p62 protein level, and induced autophagic flux. Silencing of PP2A or administration of 3-methyladenine inhibited autophagy and decreased the production of anti-inflammatory factors (IL-10, TGF-β and BDNF) and induced the release of proinflammatory cytokines (IL-1β, IL-2 and TNF-α) by STS-treated microglia, thereby inducing mitochondrial dysfunction and apoptosis of STS-treated neurons. STS exerts protection against neuron injury, and the PP2A gene plays a crucial role in improving mitochondrial function and inhibiting neuronal apoptosis by regulating autophagy and inflammation in microglia.

Downregulation of Preso protects against ischemic/reperfusion-mediated neuronal injury through regulating PSD95-nNOS/YAP pathways

Cerebral ischemic/reperfusion (I/R) injury has become a great challenge harming patients’ life. This study aims to explore the regulatory role of Preso during cerebral I/R injury and to elucidate the potential mechanism. Here, we established a middle cerebral artery occlusion/reperfusion (MCAO/IR) rat model and an oxygen-glucose deprivation/reoxygenation (OGD/R)-mediated PC12 cell model to evaluate the expression and role of Preso following cerebral I/R injury. Histopathological injury and infarct size were assessed by hematoxylin and eosin (HE) and 2,3,5-Triphenyltertrazolium chloride (TTC) staining. Double immunofluorescence staining was performed to assess neuronal apoptosis in brain tissues. Cell counting kit-8 (CCK-8) and flow cytometry were performed to evaluate cell viability and apoptosis, respectively. The reactive oxygen species (ROS) and nitric oxide (NO) levels were detected using their respective detection kits, and the expression of corresponding proteins was examined adopting Western blot. The results showed that Preso was upregulated in OGD/R-induced PC12 cells and MCAO rats. Preso knockdown significantly reduced OGD/R-caused viability loss, apoptosis and oxidative stress in PC12 cells, and reduced infarct size, attenuated histological injury, and inhibited apoptosis and oxidative stress in the brain tissues from MCAO rats, as well as inhibiting the expression of postsynaptic density protein-95 (PSD95) and nitric oxide synthase (nNOS) and repressing YAP phosphorylation in vitro. In addition, the protective role of Preso knockdown against cerebral I/R injury was partly strengthened by IC87201, the nNOS/PSD95 interaction inhibitor, or weakened by Verteporfin (Vert), an inhibitor of YAP. In conclusion, Perso knockdown might exert a protective role against cerebral I/R injury via regulating PSD95-nNOS and YAP pathways, providing a potential therapeutic target for the treatment of ischemic stroke.

Vitamin D alleviates neuronal injury in cerebral ischemia-reperfusion via enhancing the Nrf2/HO-1 antioxidant pathway to counteract NLRP3-mediated pyroptosis.

Vitamin D supplementation is reported to have anti-inflammatory and neuroprotective effects during cerebral ischemia-reperfusion injury (CIRI), but the protective mechanism has not been fully elucidated. In this study, rats were given prior administrations of 1,25-vitamin D3 (1,25-VitD3) for a week and subjected to 2 hours of middle cerebral artery occlusion (MCAO) followed by 24 hours of reperfusion. Supplementation with 1,25-VitD3 significantly reduced neurological deficit scores and cerebral infarction areas, and increased surviving neurons. Oxygen-glucose deprivation/reoxygenation (OGD/R)-induced rat cortical neuron cells (RN-C) were subjected to 1,25-VitD3 treatment. Administration of 1,25-VitD3 improved cell viability and inhibited lactate dehydrogenase (LDH) activity and cell apoptosis in OGD/R-stimulated RN-C, as assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-h-tetrazolium bromide (MTT) assay, LDH activity assays and TdT-mediated dUTP nick end labeling (TUNEL) staining, respectively. Notably, western blot assay showed that 1,25-VitD3 upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) to alleviate oxidative stress, but reduced proteins and inflammatory cytokines related to NLR pyrin domain containing 3 (NLRP3)-mediated pyroptosis, resulting in decreased pyroptosis and neuroinflammation in vivo and in vitro. Transfection of pcDNA-Nrf2 in RN-C also inhibited pyroptosis and OGD/R-induced cell death whereas breakdown of Nrf2 signals destroyed the protective effect of 1,25-VitD3 on OGD/R-stimulated RN-C. In conclusion, 1,25-VitD3 protects neurons against CIRI through activating the antioxidant Nrf2/HO-1 pathway to restrain NLRP3-mediated pyroptosis.

Dihydroartemisinin ameliorates cerebral I/R injury in rats via regulating VWF and autophagy-mediated SIRT1/FOXO1 pathway.

Dihydroartemisinin (DHA) has been found to inhibit the expression of von Willebrand factor (VWF), a marker of endothelial cell injury, but its mechanism in cerebral ischemia/reperfusion (I/R) injury remains obscure. In this study, I/R model was constructed through middle cerebral artery occlusion (MCAO) in rats, followed by DHA administration. The effect of DHA on rat cerebral I/R injury was investigated by 2,3,5-triphenyltetrazolium chloride staining, hematoxylin and eosin staining, TUNEL staining, and Western blot. Brain microvascular endothelial cells (BMVECs) isolated from newborn rats were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R), and then treated with DHA. The results showed that MCAO treatment induced infarction, nerve cell apoptosis, and brain tissue impairment in rats, which was mitigated by DHA. OGD/R inhibited viability and accelerated apoptosis of BMVECs, which was alleviated by DHA. I/R procedures or OGD/R up-regulated expressions of VWF, ATG7, Beclin1, and LC3-II/LC3-I ratio, while down-regulating Occludin, Claudin-5, ZO-1, P62, SIRT1, and FOXO1 expressions in vivo and in vitro; however, these effects of I/R procedures or OGD/R were offset by DHA. VWF overexpression reversed the above effects of DHA on OGD/R-induced BMVECs. In summary, DHA ameliorates cerebral I/R injury in rats by reducing VWF level and activating autophagy-mediated SIRT1/FOXO1 signaling pathway.

ROS-responsive exogenous functional mitochondria can rescue neural cells post-ischemic stroke.

Background: The transfer of mitochondria from healthy mesenchymal stem cells (MSCs) to injured MSCs has been shown to have potential therapeutic benefits for neural cell post-ischemic stroke. Specifically, functional mitochondria can perform their normal functions after being internalized by stressed cells, leading to host cell survival. However, while this approach shows promise, there is still a lack of understanding regarding which neural cells can internalize functional mitochondria and the regulatory mechanisms involved. To address this gap, we investigated the ability of different neural cells to internalize exogenous functional mitochondria extracted from MSCs. Methods: Functional mitochondria (F-Mito) isolated from umbilical cord derived-MSCs (UCMSCs) were labeled with lentivirus of HBLV-mito-dsred-Null-PURO vector. The ability of stressed cells to internalize F-Mito was analyzed using a mouse (C57BL/6J) middle cerebral artery occlusion (MCAO) model and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model. The cell viability was measured by CCK-8 kit. Time-course of intracellular ROS levels in stressed cells were analyzed by DCFH-DA staining after OGD/R and F-Mito treatment. MitoSOX, Mitotracker and WGA labeling were used to assess the relationship between ROS levels and the uptake of F-Mito at the single-cell level. Pharmacological modulation of ROS was performed using acetylcysteine (ROS inhibitor). Results: Our findings demonstrate that neurons and endothelial cells are more effective at internalizing mitochondria than astrocytes, both in vitro and in vivo, using an ischemia-reperfusion model. Additionally, internalized F-Mito decreases host cell reactive oxygen species (ROS) levels and rescues survival. Importantly, we found that the ROS response in stressed cells after ischemia is a crucial determinant in positively mediating the internalization of F-Mito by host cells, and inhibiting the generation of ROS chemicals in host cells may decrease the internalization of F-Mito. These results offer insight into how exogenous mitochondria rescue neural cells via ROS response in an ischemic stroke model. Overall, our study provides solid evidence for the translational application of MSC-derived mitochondria as a promising treatment for ischemic stroke.

STING mediates microglial pyroptosis via interaction with NLRP3 in cerebral ischaemic stroke

BackgroundIschaemia-evoked neuroinflammation is a critical pathogenic event following ischaemic stroke. Gasdermin D (GSDMD)-associated pyroptosis represents a type of inflammation-associated programmed cell death, which can exacerbate neuroinflammatory responses and brain damage....

Transcription factor Krüppel-like factor 4 upregulated G protein-coupled receptor 30 alleviates intestinal inflammation and apoptosis, and protects intestinal integrity from intestinal ischemia-reperfusion injury.

Intestinal ischemia/reperfusion (I/R) injury is a common clinical event occurring during multiple clinical pathological processes. Here, we designed this paper to discuss the role of G protein-coupled receptor 30 (GPR30) playing in intestinal I/R injury. An oxygen-glucose deprivation/reoxygenation (OGD/R) cell model was established to simulate the pathological process of I/R injury. With the application of enzyme-linked immunosorbent assay, TUNEL, and transepithelial electrical resistance (TEER) assays, the levels of inflammatory cytokines, cell apoptosis, and intestinal integrity were estimated. The corresponding proteins were estimated by applying western blot. Immunofluorescence was conducted to examine N-terminal Gasdermin D (GSDMD-N) expression. The interplay between KLF4 and GPR30 was demonstrated by dual-luciferase reporter assay and chromatin immunoprecipitation. The results showed that GPR30 was downregulated in Caco-2 cells exposed to OGD/R. GPR30 overexpression reduced the production of TNF-α, IL-6, IL-1β, and IL-18, the TUNEL-positive cells, as well as the contents of p-p65, Cox-2, Inos, Bax, and cleaved-PARP, but elevated the expression of Bcl-2 in OGD/R-induced Caco-2 cells. In addition, OGD/R-induced the reduction of TEER value and reduced expression of tight junction proteins in Caco-2 cells, which was partially restored by GPR30 overexpression. Furthermore, GPR30 suppressed nod-like receptor pyrin 3inflammasome and GSDMD-N expression. It was evidenced that Krüppel-like factor 4 (KLF4) could directly bind to GPR30 promoter and positively regulate GPR30 expression. The regulation of GPR30 overexpression above was weakened by KLF4 knockdown. Collectively, our findings suggested that KLF4 could transcriptionally upregulate GPR30, and GPR30 prevented intestine I/R injury by inhibiting inflammation and apoptosis, and maintaining intestinal integrity that provides potential targets for mitigating the I/R injury.