Jingxian Wu

Neuroprotection by Curcumin in Ischemic Brain Injury Involves the Akt/Nrf2 Pathway

Oxidative damage plays a critical role in many diseases of the central nervous system. This study was conducted to determine the molecular mechanisms involved in the putative anti-oxidative effects of curcumin against experimental stroke. Oxygen and glucose deprivation/reoxygenation (OGD/R) was used to mimic ischemic insult in primary cultured cortical neurons. A rapid increase in the intracellular expression of NAD(P)H: quinone oxidoreductase1 (NQO1) induced by OGD was counteracted by curcumin post-treatment, which paralleled attenuated cell injury. The reduction of phosphorylation Akt induced by OGD was restored by curcumin. Consequently, NQO1 expression and the binding activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) to antioxidant response element (ARE) were increased. LY294002 blocked the increase in phospho-Akt evoked by curcumin and abolished the associated protective effect. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion for 60 minutes. Curcumin administration significantly reduced infarct size. Curcumin also markedly reduced oxidative stress levels in middle cerebral artery occlusion (MCAO) rats; hence, these effects were all suppressed by LY294002. Taken together, these findings provide evidence that curcumin protects neurons against ischemic injury, and this neuroprotective effect involves the Akt/Nrf2 pathway. In addition, Nrf2 is involved in the neuroprotective effects of curcumin against oxidative damage.

Neuroprotection of Tanshinone IIA against Cerebral Ischemia/Reperfusion Injury through Inhibition of Macrophage Migration Inhibitory Factor in Rats

Background Ischemia/reperfusion (I/R) injury is associated with systemic inflammatory response. Macrophage migration inhibitory factor (MIF) has been implicated in many inflammatory processes. Tanshinone IIA (TSA) is one of the active ingredients in danshen, which derived from the dried root or rhizome of Salviae miltiorrhizae Bge. Recent studies have demonstrated that TSA has protective effects against focal cerebral I/R injury. However, little is known about the underlying mechanisms. Here we put forward the hypothesis that TSA acts through inhibition of MIF expression during focal cerebral I/R injury in rats. Methodology/Principal Findings Rats were subjected to middle cerebral artery occlusion (MCAO) for 2 hours. This was followed by reperfusion. We measured neurological deficits, brain water content, and infarct volume, and found that neurological dysfunction, brain edema, and brain infarction were significantly attenuated by TSA 6 hours after reperfusion. We also measured myeloperoxidase (MPO) activity at 6 and 24 hours, and found that neutrophil infiltration was significantly higher in the vehicle+I/R group than in the TSA+I/R group. ELISA demonstrated that TSA could inhibit MIF expression and the release of TNF-α and IL-6 induced by I/R injury. Western blot analysis and immunofluorescence staining showed that MIF expression was significantly lower in the TSA+I/R group than in the vehicle+I/R group. MIF was found almost all located in neurons and hardly any located in astrocytes in the cerebral cortex. Western blot analysis and EMSA demonstrated that NF-κB expression and activity were significantly increased in the vehicle+I/R group. However, these changes were attenuated by TSA. Conclusion/Significance Our results suggest that TSA helps alleviate the proinflammatory responses associated with I/R-induced injury and that this neuroprotective effect may occur through down-regulation of MIF expression in neurons.

GSK-3β downregulates Nrf2 in cultured cortical neurons and in a rat model of cerebral ischemia-reperfusion

The NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway plays a critical role in protecting against oxidative stress in brain ischemia and reperfusion injury. Glycogen synthase kinase 3β (GSK-3β) may play a critical role in regulating Nrf2 in a Kelch-like ECH-associated protein 1 (Keap1)-independent manner. However, the relationship between GSK-3β and Nrf2 in brain ischemia and reperfusion injury is not clear. In this study, we explored the mechanisms through which GSK-3β regulates Nrf2 and Nrf-2/ARE pathways in vitro and in vivo. We used oxygen and glucose deprivation/reoxygenation (OGD/R) in primary cultured cortical neurons and a middle cerebral artery occlusion-reperfusion (MCAO/R) rat model to mimic ischemic insult. In this study, GSK-3β siRNA and inhibitors (SB216763 and LiCl) were used to inhibit GSK-3β in vitro and in vivo. After inhibiting GSK-3β, expression of total and nuclear Nrf2, Nrf2-ARE binding activity, and expression of Nrf2/ARE pathway-driven genes HO-1 and NQO-1 increased. Overexpression of GSK-3β yielded opposite results. These results suggest that GSK-3β downregulates Nrf2 and the Nrf2/ARE pathway in brain ischemia and reperfusion injury. GSK-3β may be an endogenous antioxidant relevant protein, and may represent a new therapeutic target in treatment of ischemia and reperfusion injury.

Targeting thioredoxin-1 with siRNA exacerbates oxidative stress injury after cerebral ischemia/reperfusion in rats

Reactive oxygen species and their detrimental effects on the brain after transient ischemia/reperfusion (I/R) have been implicated in the pathogenesis of ischemic reperfusion injury. Thioredoxin-1 (Trx-1) is an endogenous antioxidant protein that has neuroprotective effects. We hypothesized that Trx-1 plays a crucial role in regulating cerebral I/R injury. To be able to test this, 190 Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (tMCAO) with Trx-1 siRNA (small interference RNA) injected 24 h prior to ischemia. At 24 h after tMCAO, we measured neurological deficits, infarct volume, and brain water content, and found that neurological dysfunction, brain infarct size, and brain edema were worse in the Trx-1 siRNA group than in the control group. Oxidative stress was evaluated by measuring superoxide dismutase activity and malondialdehyde level. The levels of Trx-1 and its cofactor, peroxiredoxin (Prdx), were significantly decreased after Trx-1 down-regulated. However, there is no significant difference in the Prdx mRNA level after administration of Trx-1 siRNA. In contrast, Prdx-SO3 protein levels were significantly increased in the Trx-1 siRNA group. We also investigated the specific role of nuclear factor erythroid 2-related factor 2 (Nrf2) in Trx-1 induction by knocking down Nrf2. Nrf2 siRNA injection decreased Trx-1 mRNA and protein expression. Our results suggest that the exacerbation of brain damage was associated with enhanced cerebral peroxidation in brain tissues. Moreover, these results revealed that Trx-1, which is more likely regulated by Nrf2, exerts a neuroprotective role probably through maintaining the reduction activity of Prdx1-4.

Neuroprotective effects of diallyl sulfide against transient focal cerebral ischemia via anti-apoptosis in rats

Abstract Objectives: Diallyl sulfide (DAS) is the main organosulfur component of garlic and it is known for multiple pharmacological actions. Recent studies have demonstrated that DAS has neuroprotective effects against ischemia/reperfusion injury. While some of the possible mechanisms behind this protection have been explored, its ability to inhibit apoptosis has yet to be fully explained. In the present study, the effects of DAS on focal cerebral ischemia in rats were tested and its anti-apoptotic action was explored. Methods: To examine the protective effects of DAS, focal cerebral ischemia/reperfusion was induced in rats by transient middle cerebral artery occlusion for 2 hours followed by reperfusion for 24 hours. The animals received DAS in quantities of 100, 150, and 200 mg/kg (intraperitoneal; every day), for 7 days before transient middle cerebral artery occlusion. The neurological score and infarct volume were measured at 24 hours after the end of reperfusion. Apoptotic cells were counted by terminal dUTP nick end labeling staining and apoptotic mechanisms were studied by fluorescence immunohistochemistry staining and western blot analysis. Results: For animals with induced ischemia/reperfusion, those pretreated with 200 mg/kg DAS showed an infarct volume (22·36±0·67%) significantly lower than that of the non-treated ischemia/reperfusion group (38·23±0·72%), and the percentage of terminal dUTP nick-end labeling-positive cells (23·46±1·02%) of the DAS-pretreated group was also significantly decreased compared to non-treated (36·41±1·58%). Fluorescence immunohistochemistry staining and western blot analysis indicated that DAS reduced caspase-3 expression and increased Bcl-2 expression. Conclusion: These results suggest that the mechanism by which DAS protects the brain from ischemia/reperfusion injury is related to its anti-apoptotic effects in part.

Curcumin pretreatment and post-treatment both improve the antioxidative ability of neurons with oxygen-glucose deprivation

Recent studies have shown that induced expression of endogenous antioxidative enzymes thr-ough activation of the antioxidant response element/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway may be a neuroprotective strategy. In this study, rat cerebral cortical neurons cultured in vitro were pretreated with 10 μM curcumin or post-treated with 5 μM curcumin, respectively before or after being subjected to oxygen-glucose deprivation and reoxygenation for 24 hours. Both pretreatment and post-treatment resulted in a significant decrease of cell injury as indicated by propidium iodide/Hoechst 33258 staining, a prominent increase of Nrf2 protein expression as indicated by western blot analysis, and a remarkable increase of protein expression and enzyme activity in whole cell lysates of thioredoxin before ischemia, after ischemia, and after reoxygenation. In addition, post-treatment with curcumin inhibited early DNA/RNA oxidation as indicated by immunocytochemistry and increased nuclear Nrf2 protein by inducing nuclear accumulation of Nrf2. These findings suggest that curcumin activates the expression of thioredoxin, an antioxidant protein in the Nrf2 pathway, and protects neurons from death caused by oxygen-glucose deprivation in an in vitro model of ischemia/reperfusion. We speculate that pharmacologic stimulation of antioxidant gene expression may be a promising approach to neuroprotection after cerebral ischemia.

Sulfiredoxin-1 protects PC12 cells against oxidative stress induced by hydrogen peroxide.

Oxidative stress results in protein oxidation and is implicated in cerebral disease, such as Parkinsons disease, Alzheimers disease, and ischemic stroke. Sulfiredoxin‐1 (Srxn1) is an endogenous antioxidant protein that has neuroprotective effects. The mechanisms of Srxn1 in oxidative stress have not been well studied, however. This study used 180 μM H2O2 exposure for 24 hr to model oxidative stress. This experimental design allowed us to explore the protective effects and underlying mechanisms of Srxn1 in PC12 cells. To investigate Srxn1s role in oxidative stress protection, transient knockdowns of Srxn1 in PC12 cells were performed prior to treatment with 180 μM H2O2 for 24 hr. Knockdown of Srxn1 resulted in decreased cell viability and increased cellular damage as determined by 3‐(4,5‐dimethylthiazol‐2‐yl)−2,5‐diphenyl tetrazolium bromide and lactate dehyrogenase analysis, respectively. Intracellular superoxide dismutase and glutathione are important indexes of oxidative stress; these were reduced in Srxn1 knockdown PC12. We further found that the decreased Srxn1 correlated with a reduction in 2‐Cys Prdxs activity. Moreover, 2‐Cys Prdxs protein levels were increased in the H2O2‐dosed cells, as measured by RT‐PCR and immunoblot analysis. These results suggested that Srxn1 can protect PC12 cells from H2O2‐induced oxidative stress and are involve in Prdxs activity. Srxn1 play a protective role against oxidative injury and demonstrates potential as a target for neuroprotective intervention in oxidative stress.

Sulforaphane protects primary cultures of cortical neurons against injury induced by oxygen-glucose deprivation/reoxygenation via antiapoptosis

ObjectiveTo determine whether sulforaphane (SFN) protects neurons against injury caused by oxygenglucose deprivation/reoxygenation (OGD/R) and, if so, to investigate the possible mechanisms.MethodsPrimary cultures of neurons were prepared from the cerebral cortex of 1-day-old Sprague-Dawley rats. On days 5–6 in vitro, the neurons were exposed to OGD for 1 h, followed by reoxygenation for 24 h. Cells were treated with 0, 0.1, 0.2, 0.5, 1, 2.5, or 5 μmol/L SFN, with or without 10 μmol/L LY294002, a PI3K-specific inhibitor, during OGD/R (a total of 25 h). After 24-h reoxygenation, MTT was used to assess viability and injury was assessed by Hoechst 33258/propidium iodide (PI) staining; immunofluorescence staining and Western blot were performed to detect molecular events associated with apoptosis.ResultsThe MTT assay showed that 1 μmol/L SFN significantly increased viability, and Hoechst 33258/PI staining showed that the numbers of injured neurons were reduced significantly in the SFN group. Furthermore, immunofluorescence staining and Western blot showed that SFN increased Bcl-2 and decreased cleaved caspase-3 levels. Moreover, LY294002 inhibited the phosphorylated-Akt expression evoked by SFN, decreased Bcl-2 expression and increased cleaved caspase-3 expression.ConclusionSFN protects neurons against injury from OGD/R and this effect may be partly associated with an antiapoptosis pathway.

4-Hydroxybenzyl Alcohol Confers Neuroprotection Through Up-Regulation of Antioxidant Protein Expression

An herb-derived phenolic compound, 4-hydroxybenzyl alcohol (4-HBA), exhibits beneficial effects in cerebral ischemic injury. However, the molecular mechanisms underlying this observation remain unclear. Here we used an in vitro ischemic model of oxygen–glucose deprivation followed by reperfusion (OGD/R) and an in vivo ischemic model of middle cerebral artery occlusion to investigate the relevant neuroprotective mechanisms. We demonstrated that 4-HBA reduced the neuronal injury, LDH release, and up-regulation of 8-hydroxydeoxyguanosine (8-OHdG) induced by OGD/R. Furthermore, 4-HBA reduced the cerebral infarct size and improved the behavioral parameters after cerebral ischemia. These neuroprotective effects may be conferred by the 4-HBA mediated upregulation of the transcription factor nuclear factor E2-related factor 2 (Nrf2), peroxiredoxin 6 (Prdx6) and protein disulfide isomerase (PDI) by the use of 4-HBA. Interestingly, LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocked the increase in phosphorylation of Akt and abolished the neuroprotection associated with 4-HBA. Our results suggested that 4-HBA protects neurons against cerebral ischemic injury, and this neuroprotection may occur through upregulation of Nrf2, Prdx6, and PDI expression via the PI3K/Akt pathway.

Sestrin2 Silencing Exacerbates Cerebral Ischemia/Reperfusion Injury by Decreasing Mitochondrial Biogenesis through the AMPK/PGC-1α Pathway in Rats

Sestrin2 (Sesn2) exerts neuroprotective properties in some neurodegenerative diseases. However, the role of Sesn2 in stroke is unclear. The AMP-activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1α (AMPK/PGC-1α) pathway plays an important role in regulating mitochondrial biogenesis, which helps prevent cerebral ischemia/reperfusion (I/R) injury. Here, we aimed to determine whether Sesn2 alleviated I/R damage by regulating mitochondrial biogenesis through the AMPK/PGC-1α signaling pathway. To be able to test this, Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h with Sesn2 silencing. At 24 h after reperfusion, we found that neurological deficits were exacerbated, infarct volume was enlarged, and oxidative stress and neuronal damage were greater in the Sesn2 siRNA group than in the MCAO group. To explore protective mechanisms, an AMPK activator was used. Expression levels of Sesn2, p-AMPK, PGC-1α, NRF-1, TFAM, SOD2, and UCP2 were significantly increased following cerebral I/R. However, upregulation of these proteins was prevented by Sesn2 small interfering RNA (siRNA). In contrast, activation of AMPK with 5′-aminoimidazole-4-carboxamide riboside weakened the effects of Sesn2 siRNA. These results suggest that Sesn2 silencing may suppress mitochondrial biogenesis, reduce mitochondrial biological activity, and finally aggravate cerebral I/R injury through inhibiting the AMPK/PGC-1α pathway.