Daphnetin protects hippocampal neurons from oxygen-glucose deprivation–induced injury

Daphnetin, a coumarin derivative extracted from Daphne odora var., was reported to possess a neuroprotective effect. Recently, it has been demonstrated that daphnetin attenuates ischemia/reperfusion (I/R) injury. However, the role of daphnetin in cerebral I/R injury and the potential mechanism have not been fully understood. The present study aimed to explore the regulatory roles of daphnetin on oxygen-glucose deprivation/reoxygenation (OGD/R)–induced cell injury in a model of hippocampal neurons. Our results demonstrated that daphnetin improved cell viability and reduced the lactate dehydrogenase leakage in OGD/R–stimulated hippocampal neurons. In addition, daphnetin inhibited oxidative stress and cell apoptosis in hippocampal neurons after OGD/R stimulation. Furthermore, daphnetin significantly enhanced the nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in hippocampal neurons exposed to OGD/R. Knockdown of Nrf2 blocked the protective effect of daphnetin on OGD/R–induced hippocampal neurons. In conclusion, these findings demonstrated that daphnetin attenuated oxidative stress and neuronal apoptosis after OGD/R injury through the activation of the Nrf2/HO-1 signaling pathway in hippocampal neurons. Thus, daphnetin may be a novel therapeutic agent for cerebral I/R injury.     

Higenamine reduces apoptotic cell death by induction of heme oxygenase-1 in rat myocardial ischemia-reperfusion injury

Pharmacological modulation of heme oxygenase (HO) gene expression may have significant therapeutic potential in oxidant-induced disorders, such as ischemia reperfusion (I/R) injury. Higenamine is known to reduce ischemic damages by unknown mechanism(s). The protective effect of higenamine on myocardial I/R-induced injury was investigated. Ligation of rat left anterior descending coronary artery for 30 min under anesthesia was done and followed by 24 h reperfusion before sacrifice. I/R-induced myocardial damages were associated with mitochondria-dependent apoptosis as evidenced by the increase of cytochrome c release and caspase-3 activity. Administration of higenamine (bolus, i.p) 1 h prior to I/R-injury significantly decreased the release of cytochrome c, caspase-3 activity, and Bax expression but up-regulated the expression of Bcl-2, HO-1, and HO enzyme activity in the left ventricles, which were inhibited by ZnPP IX, an enzyme inhibitor of HO-1. In addition, DNA-strand break-, immunohistochemical-analysis, and TUNEL staining also supported the anti-apoptotic effect of higenamine in I/R-injury. Most importantly, administration of ZnPP IX inhibited the beneficial effect of higenamine. Taken together, it is concluded that HO-1 plays a core role for the protective action of higenamine in I/R-induced myocardial injury.     

Glaucocalyxin B protects against oxygen-glucose-deprivation/reperfusion-induced neuronal injury in PC-12 cells

Oxidative stress has been implicated in the development of cerebral ischemia/reperfusion (I/R) injury. Glaucocalyxin B (GLB), one of five ent-kauranoid diterpenoids, was reported to possess neuroprotective activity. However, the effect of GLB on oxygen-glucose-deprivation/reperfusion (OGD/R)-induced cell injury in PC-12 cells has not been explored. PC-12 cells was treated with various concentrations of GLB (0, 2.5, 5 and 10 μM), and cell viability was detected using the MTT assay. PC-12 cells were pretreated with the indicated concentration of GLB (2.5-10 μM, 2 hours pretreatment), and were maintained under OGD for 3 hours, followed by 24 hours of reoxygenation. Cell viability was assessed using the MTT assay. The levels of superoxide dismutase, malondialdehyde, and glutathione peroxidase were detected using commercially available ELISA Kits. Intracellular reactive oxygen species level was measured using the fluorescent probe 2′,7′-dichlorofluorescein diacetate. The levels of Bcl-2, Bax, p-Akt, Akt, p-mTOR, mTOR were detected using Western blot. Our results revealed that GLB significantly protected PC12 cells against OGD/R-induced cell injury. In addition, GLB efficiently inhibited oxidative stress and cell apoptosis in OGD/R-stimulated PC-12 cells. Mechanistic studies revealed that pretreatment with GLB could induce the activation of Akt/mTOR signaling pathway resulting in protection of OGD-treated PC12 cells. In conclusion, our data indicate for the first time that GLB protects against OGD/R-induced neuronal injury in PC-12 cells. The mechanism of the protective effect of GLB is partially associated with activation of the Akt/mTOR signaling pathway. Thus, GLB may be a potential agent for protection against cerebral I/R injury.     

Overexpression of lemur tyrosine kinase-2 protects neurons from oxygen-glucose deprivation/reoxygenation-induced injury through reinforcement of Nrf2 signaling by modulating GSK-3β phosphorylation

Lemur tyrosine kinase-2 (LMTK2), a newly identified serine/threonine kinase, is a potential regulator of cell survival and apoptosis. However, little is known about its role in regulating neuronal survival during cerebral ischemia/reperfusion injury. The present study aimed to explore the potential function of LMTK2 in regulating neuronal survival using an in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury. Herein, we found that LMTK2 expression was markedly decreased in neurons following OGD/R exposure. Gain-of-function experiments demonstrated that LMTK2 overexpression significantly improved the viability and reduced apoptosis of neurons with OGD/R-induced injury. Moreover, LMTK2 overexpression reduced the production of reactive oxygen species (ROS) in OGD/R-exposed neurons. Notably, our results elucidated that LMTK2 overexpression reinforced the activation of nuclear factor erythroid 2-related factor (Nrf2)/antioxidant response element (ARE) antioxidant signaling associated with increased glycogen synthase kinase-3β (GSK-3β) phosphorylation. GSK-3β inhibition by its specific inhibitor significantly reversed LMTK2-inhibition-linked apoptosis and ROS production. Additionally, silencing Nrf2 partially reversed the LMTK2-overexpression-mediated neuroprotective effect in OGD/R-injured neurons. Taken together, our results demonstrated that LMTK2 overexpression alleviated OGD/R-induced neuronal apoptosis and oxidative damage by enhancing Nrf2/ARE antioxidant signaling via modulation of GSK-3β phosphorylation. Our study suggests LMTK2 is a potential target for neuroprotection during cerebral ischemia/reperfusion.     

Higenamine reduces HMGB1 during hypoxia-induced brain injury by induction of heme oxygenase-1 through PI3K/Akt/Nrf-2 signal pathways

Growing lines of evidence suggests that high mobility group box-1 (HMGB1) plays an important role for promoting inflammation and apoptosis in brain ischemia. Previously, we demonstrated that inducers of heme oxygenase-1 (HO-1) significantly reduce HMGB1 release in inflammatory conditions in vitro and in vivo. Thus, we tested our hypothesis that higenamine protects brain injury by inhibition of middle cerebral artery occlusion (MCAO)-mediated HMGB1 release in vivo, and glucose/glucose oxidase (GOX)-induced apoptosis in C6 cells in vitro due to HO-1 induction. Higenamine increased HO-1 expression in C6 cells in both hypoxia and normoxia, in which the former was much more significant than the latter. Higenamine increased Nrf-2 luciferase activity, translocated Nrf-2 to nucleus, and increased phosphorylation of Akt in C6 cells. Consistent with this, LY 294002, a PI3K inhibitor, inhibited HO-1 induction by higenamine and apoptosis induced by glucose/GOX in C6 cells was prevented by higenamine, which effect was reversed by LY 294002. Importantly, administration of higenamine (i.p) significantly reduced brain infarct size, mortality rate, MPO activity and tissue expression of HMGB1 in MCAO rats. In addition, recombinant high mobility group box 1 induced apoptosis in C6 cells by increasing ratio of Bax/bcl-2 and cleaved caspase c, which was inhibited by higenamine, and all of these effects were reversed by co-treatment with ZnPPIX. Therefore, we conclude that higenamine, at least in part, protects brain cells against hypoxic damages by up-regulation of HO-1. Thus, higenamine may be beneficial for the use of ischemic injuries such as stroke.     

连翘酯苷A通过Akt/Nrf2信号通路发挥抗脑缺血氧化损伤作用研究

目的:研究连翘酯苷A(Forsythiaside A,FA)对缺血再灌注引起的脑细胞损伤的保护作用及机制.方法:采用PC12细胞缺氧再复氧模型(OGD/R),细胞分组为正常组,模型组,FA处理组(1.25,2.5和5μmol/L),测定细胞存活率、凋亡率、ROS、MDA以及抗氧化酶水平.采用Western blotti...     

Suppression of REDD1 attenuates oxygen glucose deprivation/reoxygenation-evoked ischemic injury in neuron by suppressing mTOR-mediated excessive autophagy

Cerebral ischemia/reperfusion (I/R) typically occurs after mechanical thrombectomy to treat ischemic stroke, generation of reactive oxygen species (ROS) after reperfusion may result in neuronal insult, ultimately leading to disability and death. Regulated in development and DNA damage responses 1 (REDD1) is a conserved stress response protein under various pathogenic conditions. Recent research confirms the controversial role of REDD1 in injury processes. Nevertheless, the role of REDD1 in cerebral I/R remains poorly defined. In the current study, increased expression of REDD1 was observed in neurons exposed to simulated I/R via oxygen glucose deprivation/reoxygenation (OGD/R) treatment. Knockdown of REDD1 enhanced OGD/R-inhibited cell viability, but suppressed lactate dehydrogenase (LDH) release in neurons upon OGD/R. Simultaneously, suppression of REDD1 also antagonized OGD/R-evoked cell apoptosis, Bax expression, and caspase-3 activity. Intriguingly, REDD1 depression abrogated neuronal oxidative stress under OGD/R condition by suppressing ROS, MDA generation, and increasing antioxidant SOD levels. Further mechanism analysis corroborated the excessive activation of autophagy in neurons upon OGD/R with increased expression of autophagy-related LC3 and Beclin-1, but decreased autophagy substrate p62 expression. Notably, REDD1 inhibition reversed OGD/R-triggered excessive neuronal autophagy. More importantly, depression of REDD1 also elevated the expression of p-mTOR. Preconditioning with mTOR inhibitor rapamycin engendered not only a reduction in mTOR activation, but also a reactivation of autophagy in REDD1 knockdown-neurons upon OGD/R. In addition, blocking the mTOR pathway muted the protective roles of REDD1 inhibition against OGD/R-induced neuron injury and oxidative stress. Together these data suggested that REDD1 may regulate I/R-induced oxidative stress injury in neurons by mediating mTOR-autophagy signaling, supporting a promising therapeutic strategy against brain ischemic diseases.     

Knockdown of TRIM32 Protects Hippocampal Neurons from Oxygen–Glucose Deprivation-Induced Injury

Tripartite motif 32 (TRIM32) is a member of TRIM family that plays a potential role in neural regeneration. However, the biological function of TRIM32 in cerebral ischemia reperfusion injury has not been investigated. In the present study, we evaluated the expression level of TRIM32 in hippocampal neurons following oxygen–glucose deprivation/reperfusion (OGD/R). The results showed that TRIM32 expression was significantly elevated in hippocampal neurons subjected to OGD/R as compared to the neurons cultured in the normoxia condition. To further evaluate the role of TRIM32, hippocampal neurons were transfected with TRIM32 small interfering RNA (si-TRIM32) to knock down TRIM32. We found that knockdown of TRIM32 improved cell viability of OGD/R-stimulated hippocampal neurons. Generation of reactive oxygen species was decreased, while contents of superoxide dismutase and glutathione peroxidase were increased after si-TRIM32 transfection. Knockdown of TRIM32 suppressed cell apoptosis, as proved by the increased bcl-2 expression along with decreased bax expression and caspase-3 activity. We also found that TRIM32 knockdown enhanced OGD/R-induced activation of Nrf2 signaling pathway in hippocampal neurons. Furthermore, siRNA-Nrf2 was transfected to knock down Nrf2. SiRNA-Nrf2 transfection reversed the protective effects of TRIM32 knockdown on neurons. These data suggested that knockdown of TRIM32 protected hippocampal neurons from OGD/R-induced oxidative injury through activating Nrf2 signaling pathway.

     

Activation of the Nrf2/HO-1 Antioxidant Pathway Contributes to the Protective Effects of Lycium Barbarum Polysaccharides in the Rodent Retina after Ischemia-Reperfusion-Induced Damage

Lycium barbarum polysaccharides (LBP), extracts from the wolfberries, are protective to retina after ischemia-reperfusion (I/R). The antioxidant response element (ARE)–mediated antioxidant pathway plays an important role in maintaining the redox status of the retina. Heme oxygenase-1 (HO-1), combined with potent AREs in its promoter, is a highly effective therapeutic target for the protection against neurodegenerative diseases, including I/R-induced retinal damage. The aim of our present study was to investigate whether the protective effect of LBP after I/R damage was mediated via activation of the Nrf2/HO-1-antioxidant pathway in the retina. Retinal I/R was induced by an increase in intraocular pressure to 130 mm Hg for 60 minutes. Prior to the induction of ischemia, rats were orally treated with either vehicle (PBS) or LBP (1 mg/kg) once a day for 1 week. For specific experiments, zinc protoporphyrin (ZnPP, 20 mg/kg), an HO-1 inhibitor, was intraperitoneally administered at 24 h prior to ischemia. The protective effects of LBP were evaluated by quantifying ganglion cell and amacrine cell survival, and by measuring cell apoptosis in the retinal layers. In addition, HO-1 expression was examined using Western blotting and immunofluorescence analyses. Cytosolic and nuclear Nrf2 was measured using immunofluorescent staining. LBP treatment significantly increased Nrf2 nuclear accumulation and HO-1 expression in the retina after I/R injury. Increased apoptosis and a decrease in the number of viable cells were observed in the ganglion cell layer (GCL) and inner nuclear layer (INL) in the I/R retina, which were reversed by LBP treatment. The HO-1 inhibitor, ZnPP, diminished the LBP treatment-induced protective effects in the retina after I/R. Taken together, these results suggested that LBP partially exerted its beneficial neuroprotective effects via the activation of Nrf2 and an increase in HO-1 protein expression.     

Platycodin D protects cortical neurons against oxygen-glucose deprivation/reperfusion in neonatal hypoxic-ischemic encephalopathy

Neonatal hypoxic-ischemic encephalopathy is one of the leading causes of death in infants. Increasing evidence indicates that oxidative stress and apoptosis are major contributors to hypoxic-ischemic injury and can be used as particularly promising therapeutic targets. Platycodin D (PLD) is a triterpenoid saponin that exhibits antioxidant properties. The aim of this study was to evaluate the effects of PLD on hypoxic-ischemic injury in primary cortical neurons. We found that oxygen-glucose deprivation/reperfusion (OGD/R) induced inhibition of cell viability and cytotoxicity, which were attenuated by PLD treatment. PLD treatment inhibited oxidative stress induced by OGD/R, which was evidenced by the reduced level of reactive oxygen species and increased activities of catalase, superoxide dismutase, and glutathione peroxidase. Histone-DNA enzyme-linked immunosorbent assay revealed that apoptosis was significantly decreased after PLD treatment in OGD/R-treated cortical neurons. The increased bax expression and decreased bcl-2 expression induced by OGD/R were reversed by PLD treatment. Furthermore, PLD treatment caused the activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in OGD/R-stimulated cortical neurons. Suppression of this pathway blocked the protective effects of PLD on OGD/R-induced cell injury. These findings suggested that PLD executes its protective effects on OGD/R-induced cell injury via regulating the PI3K/Akt/mTOR pathway in cortical neurons.     

Phytic acid exerts protective effects in cerebral ischemia-reperfusion injury by activating the anti-oxidative protein sestrin2

ABSTRACT

Cerebral ischemia reperfusion (I/R) is a therapeutic strategy for ischemia; however, it usually causes injury by the aspect of inflammation and neuron apoptosis. This investigation aims to investigate the protective effects of phytic acid (IP6) for cerebral I/R injury in vitro. PC-12 cells under Oxygen and glucose deprivation/reperfusion (OGD/R) were performed to mimic cerebral I/R. IP6 was pretreated before PC-12 cells under OGD/R treatment. The data showed that IP6 activated the expression of sestrin2 in OGD/R injured PC-12 cells. IP6 inhibited OGD/R induced inflammation, oxidative stress, and apoptosis by activating sestrin2. Besides, p38 MAPK may mediate the effects of sestrin2 activated by IP6. Therefore, IP6 can be a potential drug to prevent neurological damage in cerebral I/R injury.     

Cardiomyocyte protective effects of thyroid hormone during hypoxia/reoxygenation injury through activating of IGF-1-mediated PI3K/Akt signalling

Ischaemia/reperfusion (I/R) injury is a common clinical condition that results in apoptosis and oxidative stress injury. Thyroid hormone was previously reported to elicit cardiac myocyte hypertrophy and promote cardiac function after cardiac injury. We used an in vivo mouse model of I/R injury and in vitro primary cardiomyocyte culture assays to investigate the effects of thyroid hormone on cardiomyocytes during hypoxia/reoxygenation (H/R) injury. The results showed that T3 pretreatment in vivo significantly improved left ventricular function after I/R injury. In vitro, T3 pretreatment decreased cell apoptosis rate, inhibited caspase-3 activity and decreased the Bax/Bcl-2 ration induced by H/R injury. T3 pretreatment significantly attenuated the loss of mitochondrial membrane potential. Furthermore, it was observed that T3 diminished the expression of NCX1 protein and decreased SERCA2a protein expression in H/R-induced cardiomyocytes, and T3 prevented intracellular Ca²⁺ increase during H/R injury. Also, T3 increased the expression of IGF-1, and PI3K/Akt signalling in cardiomyocytes under H/R-induced injury, and that the protective effect of T3 against H/R-induced injury was blocked by the PI3K inhibitor LY294002. IGF-1 receptor (IGF-1R) inhibitor GSK1904529A significantly inhibited the expression of IGF-1R and PI3K/Akt signalling. In summary, T3 pretreatment protects cardiomyocytes against H/R-induced injury by activating the IGF-1-mediated PI3K/Akt signalling pathway.     

Sertad1 Induces Neurological Injury after Ischemic Stroke via the CDK4/p-Rb Pathway

SERTA domain-containing protein 1 (Sertad1) is upregulated in the models of DNA damage and Alzheimer’s disease, contributing to neuronal death. However, the role and mechanism of Sertad1 in ischemic/hypoxic neurological injury remain unclear. In the present study, our results showed that the expression of Sertad1 was upregulated in a mouse middle cerebral artery occlusion and reperfusion model and in HT22 cells after oxygen-glucose deprivation/reoxygenation (OGD/R). Sertad1 knockdown significantly ameliorated ischemia-induced brain infarct volume, neurological deficits and neuronal apoptosis. In addition, it significantly ameliorated the OGD/R-induced inhibition of cell viability and apoptotic cell death in HT22 cells. Sertad1 knockdown significantly inhibited the ischemic/hypoxic-induced expression of p-Rb, B-Myb, and Bim in vivo and in vitro. However, Sertad1 overexpression significantly exacerbated the OGD/R-induced inhibition of cell viability and apoptotic cell death and p-Rb, B-Myb, and Bim expression in HT22 cells. In further studies, we demonstrated that Sertad1 directly binds to CDK4 and the CDK4 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"ON123300","term_id":"2214659616"}}ON123300 restores the effects of Sertad1 overexpression on OGD/R-induced apoptotic cell death and p-Rb, B-Myb, and Bim expression in HT22 cells. These results suggested that Sertad1 contributed to ischemic/hypoxic neurological injury by activating the CDK4/p-Rb pathway.     

Nitro-oleic acid ameliorates oxygen and glucose deprivation/re-oxygenation triggered oxidative stress in renal tubular cells via activation of Nrf2 and suppression of NADPH oxidase

Nitroalkene derivative of oleic acid (OA-NO₂), due to its ability to mediate revisable Michael addition, has been demonstrated to have various biological properties and become a therapeutic agent in various diseases. Though its antioxidant properties have been reported in different models of acute kidney injury (AKI), the mechanism by which OA-NO₂ attenuates intracellular oxidative stress is not well investigated. Here, we elucidated the anti-oxidative mechanism of OA-NO₂ in an in vitro model of renal ischemia/reperfusion (I/R) injury. Human tubular epithelial cells were subjected to oxygen and glucose deprivation/re-oxygenation (OGD/R) injury. Pretreatment with OA-NO₂ (1.25?μM, 45?min) attenuated OGD/R triggered reactive oxygen species (ROS) generation and subsequent mitochondrial membrane potential disruption. This action was mediated via up-regulating endogenous antioxidant defense components including superoxide dismutase (SOD1), heme oxygenase 1 (HO-1), and γ-glutamyl cysteine ligase modulatory subunits (GCLM). Moreover, subcellular fractionation analyses demonstrated that OA-NO₂ promoted nuclear translocation of nuclear factor-E2- related factor-2 (Nrf2) and Nrf2 siRNA partially abrogated these protective effects. In addition, OA-NO₂ inhibited NADPH oxidase activation and NADPH oxidase 4 (NOX4), NADPH oxidase 2 (NOX2) and p22^phox up-regulation after OGD/R injury, which was not relevant to Nrf2. These results contribute to clarify that the mechanism of OA-NO₂ reno-protection involves both inhibition of NADPH oxidase activity and induction of SOD1, Nrf2-dependent HO-1, and GCLM.