ngyu Li

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.

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.

Inhibition of thioredoxin-1 with siRNA exacerbates apoptosis by activating the ASK1-JNK/p38 pathway in brain of a stroke model rats.

Apoptosis is critical for the development of cerebral ischemia/reperfusion injury. Thioredoxin-1(Trx-1) protein has been reported to have anti-apoptotic effects in a variety of cell types, and it has been implicated in brain injury after middle cerebral artery occlusion (MCAO). Thus, we studied the effects of Trx1 silencing after MCAO in rats and examined whether inhibition of endogenous Trx1 could increase tissue levels of apoptosis. Male Sprague-Dawley rats (N=170) were subjected to 1h of middle cerebral arterial occlusion followed by 24h of reperfusion. Trx1 siRNAs were injected into rat brains 24h prior to MCAO. Then, 24h after MCAO, brains were collected from euthanized rats for investigation. Treatment with Trx1 siRNA significantly increased mortality, behavioral deficits, and cerebral infarction volume and exacerbated neuronal cell apoptotic death after MCAO injury. Western blot revealed increased expression of apoptotic proteins such as P-ASK1, P-JNK, P-p38, cleaved caspase-3 and increased the level of cytochrome c in the cytosolic fraction in the Trx1 siRNA-treated group. Co-immunoprecipitation assay suggested an interaction between Trx1 and ASK1 in normal rat brains and Trx1 siRNA dissociated ASK1-Trx1 binding complex. Our data suggest that Trx1 siRNA increases apoptotic stress-induced ASK1 activation and this represents further evidence that Trx1 is an endogenous anti-apoptotic molecule that diminishes focal cerebral ischemia/reperfusion injury. Its mechanism of action is likely related to attenuation of the ASK1-JNK/p38 signaling pathway.

Resveratrol alleviates cerebral ischemia/reperfusion injury in rats by inhibiting NLRP3 inflammasome activation through Sirt1-dependent autophagy induction.

Abstract Resveratrol has been reported to protect against cerebral ischemia/reperfusion (I/R) injury in rats, but the underlying mechanism is unclear. In the current study, we examined whether resveratrol ameliorates cerebral I/R injury by inhibiting NLRP3 inflammasome‐derived inflammation and whether autophagy is involved in this process. In addition, we explored the role of Sirt1 in resveratrol‐mediated protective effects. To answer these questions, healthy male Sprague‐Dawley rats were exposed to middle cerebral artery occlusion for 1 h followed by 24 h reperfusion. We found that cerebral I/R increased levels of activated NLRP3 inflammasome, caspase‐1, IL‐1&bgr;, and IL‐18 and enhanced autophagy activity (ratio of LC3B‐II/LC3B‐I and p62/SQSTM1). Treatment with resveratrol, a specific Sirt1 agonist, attenuated I/R‐induced NLRP3 inflammasome‐derived inflammation but upregulated autophagy. Furthermore, resveratrol treatment clearly reduced cerebral infarct volume, decreased brain water content, and improved neurological scores. In addition, inhibition of autophagy using 3‐MA intracerebroventricular injection blocked the inhibitory effect of resveratrol on NLRP3 inflammasome activation. Finally, Sirt1 knockdown with siRNA significantly blocked resveratrol‐induced enhancement of autophagy activity and suppression of NLRP3 inflammasome activation. In conclusion, our results demonstrate that resveratrol protects against cerebral I/R injury by inhibiting NLRP3 inflammasome activation through Sirt1‐dependent autophagy activity. HighlightsResveratrol treatment alleviates cerebral ischemia/reperfusion injury by inhibiting NLRP3 inflammasome activationResveratrol suppresses NLRP3 inflammasome activation by increasing autophagy activitySirt1 plays a vital role in resveratrol‐mediated enhancement of autophagy activitySirt1‐dependent autophagy activity is critical for resveratrol‐mediated inhibition of NLRP3 inflammasome activation

Nrf2 inhibits NLRP3 inflammasome activation through regulating Trx1/TXNIP complex in cerebral ischemia reperfusion injury

The nod-like receptor protein 3 (NLRP3) inflammasome has a critical role in inflammation damage in ischemic injury, and the activation of the inflammasome is closely related to the interaction with thioredoxin interacting protein (TXNIP), which dissociates from the thioredoxin1 (Trx1)/TXNIP complex under oxidative stress. However, the negative regulator of NLRP3 inflammasome activation has not been fully investigated. Nuclear factor erythroid 2-related factor 2 (Nrf2) takes on a critical part in the antioxidant stress system, that controls the driven genes of antioxidant response element (ARE). Activate Nrf2 could inhibit the activation of NLRP3 inflammasome in acute liver injury and severe lupus nephritis. We aimed to explore the protective effect of Nrf2 in inhibiting the NLPR3 inflammasome formulation through the Trx1/TXNIP complex in cerebral ischemia reperfusion (cerebral I/R) injury. Middle cerebral artery occlusion/reperfusion (MCAO/R) model was used to imitate ischemic insult. Nrf2 was activated by tert-butylhydroquinone (tBHQ) intraperitoneally (i.p.) injection (16.7mg/kg), Nrf2,Trx1 and NLRP3 siRNAs were infused into the left paracele (12μl per rat), protein and mRNA levels were assessed by Western blot, qRT-PCR. ELISA was used for IL-1β and IL-18 activity measurements. After upregulating Nrf2, the expression of TXNIP in cytoplasm, NLRP3 inflammasome, and downstream factors caspase-1, IL-18, and IL-1β were significantly reduced, and Nrf2 knockdown yielded the opposite results. Trx1 knockdown produced the same effect of Nrf2 inhibition and the protective effect of Nrf2 was mostly abolished. Our results suggested that Nrf2 acted as a protective regulator against NLRP3 inflammasome activation by regulating the Trx1/TXNIP complex, which could possibly represent an innovative insight into the treatment of ischemia and reperfusion injury.

Glycogen Synthase Kinase 3β Influences Injury Following Cerebral Ischemia/Reperfusion in Rats

Abnormal activation of GSK-3β is associated with psychiatric and neurodegenerative disorders. However, no study has examined the effect of GSK-3β on cerebral ischemia/reperfusion injury. We used oxygen-glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion (MCAO) as models of ischemia/reperfusion in rats in vitro and in vivo. Our study showed that knockdown of GSK-3β with a GSK-3β siRNA virus improved injury and increased viability of neurons subjected to OGD/R. Levels of total Nrf2, nuclear Nrf2, and Nrf2 downstream proteins sulfiredoxin (Srx1) and thioredoxin (Trx1) increased after transfection with the GSK-3β siRNA virus. GSK-3β siRNA increased SOD activity and decreased MDA levels. Overexpression of GSK-3β with a pcDNA-GSK-3β virus showed opposite results. We also demonstrated that intracerebroventricular injection of GSK-3β siRNA in rats ameliorated neurological deficits, reduced brain infarct volume and water content, and reduced damage to cerebral cortical neurons after MCAO. Changes in total Nrf2, nuclear Nrf2, Srx1, Trx1, SOD, and MDA were similar to those observed in vitro. Our results show for the first time that GSK-3β can influence cerebral ischemia/reperfusion injury. The effects may be due to regulating the Nrf2/ARE pathway and decreasing oxidative stress. These results suggest a potential new drug target for clinical treatment of stroke.

Neuroprotective effects of sulfiredoxin-1 during cerebral ischemia/reperfusion oxidative stress injury in rats

As an endogenous antioxidant protein, Sulfiredoxin1 (Srxn1) can prevent cell oxidative stress damage. However, its role in cerebral ischemia/reperfusion (I/R) injury and the underlying signaling mechanisms remain largely unknown. Here, we explored effects of Srxn1 knockdown on oxidative stress using in vitro and in vivo I/R models and investigated related neuroprotective mechanisms. For in vitro studies, primary cortical neuronal cultures were transfected with an interfering lentivirus targeting Srxn1. Oxygen-glucose deprivation (OGD) was conducted after Srxn1 knockdown. MTS and lactate dehydrogenase assays indicated that knockdown of Srxn1 increased cell death and reduced cell viability. Similarly, superoxide dismutase (SOD) and reduced glutathionekits assays showed that knockdown of Srxn1 worsened oxidative stress injury. For in vivo studies, siRNA for Srxn1 or negative control siRNA was injected intracerebroventricularly 24h before middle cerebral artery occlusion (MCAO). Data shows silencing Srxn1 resulted in a significant increase in cerebral infarction, neurological deficits, histological injury, and oxidative stress injury 24h after ischemic stroke. Moreover, immunoblot analysis assessed the relationship between Srxn1 levels and Prdx1-4 as well as Prdx-SO3 activity both in vitro and in vivo models. We found that decreased Srxn1 reduced Prdx1-4 and enhanced Prdx-SO3 protein levels. In addition, knockdown of Nrf2 was performed; immunoblot analysis was used to measure Srxn1 and NQO1 protein levels. We further found that interference of Nrf2 reduced Srxn1 and NQO1 protein levels. In summary, Srxn1 can protect neurons from I/R oxidative stress injury and the mechanism involves Prdx activity. Srxn1, which might be downstream of Nrf2, can prevent cerebral ischemia reperfusion by reversing overoxidized Prdx and restoring antioxidant activity of Prdx.

Thioredoxin 1 protects astrocytes from oxidative stress by maintaining peroxiredoxin activity

Previous studies have demonstrated that thioredoxin 1 (Trx1) exerts neuroprotective effects against cerebral ischemia/reperfusion injury caused by oxidative stress. While Trx1 is known to maintain the anti-oxidant activity of 2-Cys peroxiredoxins (Prdxs), the underlying mechanisms of its protective effects have remained to be elucidated, which was the aim of the present study. For this, an in vitro ischemic model of hypoxemia lasting for 4 h, followed by 24 h of reperfusion was used. Primary astrocytes from neonatal rats were pre-treated with small interfering RNA targeting Trx1 prior to oxygen glucose deprivation/reperfusion (OGD/R). MTS and lactate dehydrogenase assays were performed to evaluate cell viability. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were employed to assess the mRNA and protein expression levels of Prdx1-4 and Prdx-SO3. Furthermore, a dual luciferase reporter assay was used to assess the interaction between activator protein-1 (AP-1) and Trx1. The present study demonstrated that OGD/R decreased the cell viability and increased cellular damage, which was more marked following Trx1 knockdown. The expression of Prdx1-4 and Prdx-SO3 protein was higher in the cells subjected to OGD/R. Knockdown of Trx1 markedly decreased the levels of Prdx1-4 but increased Prdx-SO3 mRNA and protein levels. The results of the present study also suggested that AP-1 directly activated the expression of Trx1. The present study demonstrated that Trx1 exerts its neuroprotective effects by preventing oxidative stress in astrocytes via maintaining Prdx expression.

C1q/Tumor Necrosis Factor-Related Protein-3 Attenuates Brain Injury after Intracerebral Hemorrhage via AMPK-Dependent Pathway in Rat.

C1q/tumor necrosis factor (TNF)-related protein-3 (CTRP3) is a recently discovered adiponectin paralog with established metabolic regulatory properties. However, the role of CTRP3 in intracerebral hemorrhage (ICH) is still mostly unresolved. The aim of the present report was to explore the possible neuroprotective effect of CTRP3 in an ICH rat model and to elucidate the fundamental mechanisms. ICH was induced in rats by intracerebral infusion of autologous arterial blood. The effects of exogenous CTRP3 (recombinant or lentivirus CTRP3) on brain injury were explored on day 7. Treatment with CTRP3 reduced brain edema, protected against disruption of the blood-brain barrier (BBB), improved neurological functions and promoted angiogenesis. Furthermore, CTRP3 greatly intensified phosphorylation of AMP-activated protein kinase (AMPK) in addition to expression of hypoxia inducing factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Finally, the protective effects of CTRP3 could be blocked by either AMPK or VEGF inhibitors. Our findings give the first evidence that CTRP3 is a new proangiogenic and neuroprotective adipokine, which may exert its protective effects at least partly through an AMPK/HIF-1α/ VEGF-dependent pathway, and suggest that CTRP3 may provide a new therapeutic strategy for ICH.