Down-Regulation of miRNA-30a Alleviates Cerebral Ischemic Injury Through Enhancing Beclin 1-Mediated Autophagy

The understanding of molecular mechanism underlying ischemia/reperfusion-induced neuronal death and neurological dysfunction may provide therapeutic targets for ischemic stroke. The up-regulated miRNA-30a among our previous identified 19 MicroRNAs (miRNAs) in mouse brain after 6 h middle cerebral artery occlusion (MCAO) could negatively regulate Beclin 1 messenger RNA (mRNA) resulting in decreased autophagic activity in tumor cells and cardiomyocytes, but its role in ischemic stroke is unclear. In this study, the effects of miRNA-30a on ischemic injury in N2A cells and cultured cortical neurons after oxygen glucose deprivation (OGD), and mouse brain with MCAO-induced ischemic stroke were evaluated. The results showed that miRNA-30a expression levels were up regulated in the brain of mice after 6 h MCAO without reperfusion, but significantly down regulated in the peri-infarct region of mice with 1 h MCAO/24 h reperfusion and in N2A cells after 1 h OGD/6–48 h reoxygenation. Both the conversion ratio of microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I and Beclin 1 protein level increased in N2A cells and cultured cortical neurons following 1 h OGD/24 h reoxygenation. The down-regulated miRNA-30a could attenuate 1 h OGD/24 h reoxygenation-induced ischemic injury in N2A cells and cultured cortical neurons through enhancing Beclin 1-mediated autophagy, as miRNA-30a recognized the 3′-untranslated region of beclin 1 mRNA to negatively regulate Beclin 1-protein level via promoting beclin 1 messenger RNA (mRNA) degradation, and Beclin 1 siRNA abolished anti-miR-30a-induced neuroprotection in 1 h OGD/24 h reoxygenation treated N2A cells. In addition, anti-miR-30a attenuated the neural cell loss and improved behavioral outcome of mice with ischemic stroke. These results suggested that down-regulation of miRNA-30a alleviates ischemic injury through enhancing beclin 1-mediated autophagy, providing a potential therapeutic target for ischemic stroke.     

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.     

Analyses of changes in myocardial long non-coding RNA and mRNA profiles after severe hemorrhagic shock and resuscitation via RNA sequencing in a rat model

Background

Ischemia–reperfusion injury has been proven to induce organ dysfunction and death, although the mechanism is not fully understood. Long non-coding RNAs (lncRNAs) have drawn wide attention with their important roles in the gene expression of some biological processes and diseases, including myocardial ischemia–reperfusion (I/R) injury. In this paper, a total of 26 Sprague–Dawley (SD) rats were randomized into two groups: sham and ischemia–reperfusion (I/R) injury. Hemorrhagic shock was induced by removing 45% of the estimated total blood volume followed by reinfusion of shed blood. High-throughput RNA sequencing was used to analyze differentially expressed (DE) lncRNAs and messenger RNAs (mRNAs) in the heart tissue 4 h after reperfusion. Myocardial function was also evaluated.

Results

After resuscitation, the decline of myocardial function of shocked animals, expressed by cardiac output, ejection fraction, and myocardial performance index (MPI), was significant (p?<?0.05). DE lncRNAs and mRNAs were identified by absolute value of fold change?≥?2 and the false discovery rate ≤?0.001. In rats from the I/R injury group, 851 lncRNAs and 1015 mRNAs were significantly up-regulated while 1533 lncRNAs and 1702 m RNAs were significantly down-regulated when compared to the sham group. Among the DE lncRNAs, we found 12 location-associated with some known apoptosis-related protein-coding genes which were up-regulated or down-regulated accordingly, including STAT3 and Il1r1. Real time PCR assays confirmed that the expression levels of five location-associated lncRNAs (NONRATT006032.2, NONRATT006033.2, NONRATT006034.2, NONRATT006035.2 and NONRATT029969.2) and their location-associated mRNAs (STAT3 and Il1r1) in the rats from the I/R injury group were all significantly up-regulated versus the sham group.

Conclusions

The DE lncRNAs (NONRATT006032.2, NONRATT006033.2, NONRATT006034.2 and NONRATT006035.2) could be compatible with their role in myocardial protection by stimulating their co-located gene (STAT3) after hemorrhagic shock and resuscitation. The final prognosis of I/R injury might be regulated by different genes, which is regarded as a complex network.

     

Cover Image,Volume 234, Number 8, August 2019

KCNQ/M potassium channels play a vital role in neuronal excitability; however, it is required to explore their pharmacological modulation on N-Methyl- d -aspartic acid receptors (NMDARs)-mediated glutamatergic transmission of neurons upon ischemic insults. In the current study, both presynaptic glutamatergic release and activities of NMDARs were measured by NMDAR-induced miniature excitatory postsynaptic currents (mEPSCs) in cultured cortical neurons of C57 mice undergoing oxygen and glucose deprivation (OGD) or OGD/reperfusion (OGD/R). The KCNQ/M-channel opener, retigabine (RTG), suppressed the overactivation of postsynaptic NMDARs induced by OGD and then NO transient; RTG also decreased OGD-induced neuronal death measured with MTT assay, suggesting the beneficial role of KCNQ/M-channels for the neurons exposed to ischemic insults. However, when the neurons exposed to the subsequent reperfusion, KCNQ/M-channels played a differential role from its protective effect. OGD/R increased presynaptic glutamatergic release, which was further augmented by RTG or decreased by KCNQ/M-channel blocker, XE991. Reactive oxygen species (ROS) were produced partly in a NO-dependent manner. In addition, XE991 decreased neuronal injuries upon reperfusion measured with DCF and PI staining. Meanwhile, the addition of RTG upon OGD or XE991 upon reperfusion can reverse OGD or OGD/R-reduced mitochondrial membrane potential. Our present study indicates the dual role of KCNQ/M-channels in OGD and OGD/R, which will decide the fate of neurons. Provided that activation of KCNQ/M-channels has differential effects on neuronal injuries during OGD or OGD/R, we propose that therapy targeting KCNQ/M-channels may be effective for ischemic injuries but the proper timing is so crucial for the corresponding treatment.     

Long non-coding RNA RMST silencing protects against middle cerebral artery occlusion (MCAO)-induced ischemic stroke

Long non-coding RNAs (lncRNAs) have emerged as major regulators in neurological diseases, and clarifying their roles in cerebral ischemic injury may provide novel targets for treating ischemic stroke. In this study, we mainly studied the role of lncRNA-RMST in middle cerebral artery occlusion (MCAO)-induced mouse brain injury. We showed that RMST expression level was significantly up-regulated in oxygen-glucose deprivation (OGD)-treated primary hippocampal neuron, MCAO-induced injured brain, and the plasma of patients with ischemic stroke. RMST silencing protected against MCAO-induced ischemic brain injury in vivo and OGD-induced primary hippocampal neuron injury in vitro. Intracerebroventricular injection of RMST shRNA significantly decreased brain RMST expression, reduced brain infarct size, and improved neurological function. Collectively, this study provides evidence that lncRNA is involved in the pathogenesis of ischemic brain injury, and suggests a promising approach of RMST inhibition in treating ischemic stroke.     

Neuritin Attenuates Neuronal Apoptosis Mediated by Endoplasmic Reticulum Stress In Vitro

Neuritin is an extracellular glycophosphatidylinositol-linked protein that promotes neuronal survival, differentiation, function, and repair, but the exact mechanism of this neuroprotective effect remains unclear. Meanwhile, endoplasmic reticulum stress (ERS) induced apoptosis is attracting increased attention. In this work, we hypothesized that neuritin inhibited ERS to protect cortical neurons. To check this hypothesis, we exposed primary cultured cortical neurons to oxygen and glucose deprivation (OGD) for 45 min followed by reperfusion (R) to activate ERS. We then performed resuscitation for 6, 12, 24, and 48 h. ERS-related factors such as glucose-regulated protein 78 (GRP78), caspase-12 and CHOP were detected by Western blotting and quantitative real-time polymerase chain reaction assay. Apoptosis was assessed by Annexin V binding and propidium iodide staining. Ultrastructural changes of endoplasmic reticulum were observed under a transmission electron microscope. Results showed that GRP78 expression significantly increased at 12, 24, and 48 h and peaked at 24 h. Caspase-12 and CHOP expression significantly increased in a time-dependent manner at 12, 24, and 48 h. GRP78, caspase-12 and CHOP expression as well as apoptosis rate of primary cultured neurons and the ultrastructural changes of endoplasmic reticulum in the OGD/R?+?neuritin group significantly improved compared with the OGD/R group. In conclusion, the neuroprotection function of neuritin may be involved in ERS pathways.     

Progesterone Attenuates Aquaporin-4 Expression in an Astrocyte Model of Ischemia/Reperfusion

Previous studies have suggested that progesterone may be involved in neuroprotection by preventing brain edema. In this study, we assessed the effects of progesterone on aquaporin-4 (AQP4) expression in an ischemia/reperfusion model of cultured rat astrocytes, and further explored the possible role of the protein kinase C (PKC) pathway in this course. We evaluate primary culture astrocytes exposed to 4 h oxygen–glucose deprivation (OGD) followed by 24 h reperfusion (OGD4h/R24h) as a means of simulating cortex ischemia and reperfusion, and test the effect of progesterone on AQP4 expression in response to OGD4h/R24h. Besides, the cell viability was assessed by MTT reduction and lactate dehydrogenase release assay, accompanied by cell morphology survey. At a concentration of 1 and 2 μM, progesterone significantly attenuated AQP4 at the level of both protein and mRNA and ameliorated the cell viability of astrocytes from OGD/reperfusion injury. Moreover, this effect was blocked by the PKC inhibitor Ro31-8220, which was employed before the OGD. These results indicate that progesterone exerts the protective effects and attenuates AQP4 expression in an astrocyte model of ischemia/reperfusion depending on the PKC signal pathway.     

δ-联蛋白通过AKT信号通路调控OGD/R后的炎症反应

δ-联蛋白(delta-catenin)作为高表达于神经系统的黏附蛋白质,在神经系统的功能发挥中有着至关重要的作用,但其在缺血缺氧性脑病中的研究尚未见报道。本文通过体外培养原代皮层神经元,构建氧糖剥夺/再灌注（oxygen-glucose deprivation/reperfusion, OGD/R）模型。用Western 印迹、LDH等方法显示,与对照组相比,δ-联蛋白在OGD/R模型后的不同时间点（0、4、12、24 和48 h）表达呈先降低后升高的趋势。在12 h表达量最低（0.48±0.08 vs 1.53±0.18,P<0.05）,在48 h表达升高到对照组水平（1.35±0.15 vs 1.53±0.18,P>0.05）。用siRNA慢病毒干扰δ-联蛋白表达后,ELISA结果显示,和对照组相比,OGD/R后,IL-1β和IL-18升高（24.80±1.64 vs 12.75±0.87,28.12±2.69 vs 12.99±1.24,P<0.05）,但在干扰δ-联蛋白表达后,和OGD组相比,IL-1β和IL-18释放降低（12.81±0.78 vs 24.80±1.64,14.27±1.37 vs 28.12±2.69,P<0.05）。Western 印迹结果显示,AKT信号通路磷酸化位点Ser 473活化增高（1.08±0.04 vs 0.85±0.06,P<0.05）,但Thr 308位点活化无改变（1.17±0.06 vs 1.11±0.08,P>0.05）。在siRNA慢病毒干扰并且联合使用AKT信号通路抑制剂GSK 690693后,和OGD+siRNA组相比,IL-1β和IL-18释放增高（24.58±0.99 vs 12.81±0.78,31.62±2.23 vs 14.27±1.37,P<0.05）。上述结果显示,δ 联蛋白通过AKT信号通路调控OGD/R后的炎症反应,这可作为δ-联蛋白功能研究的新的实验依据。     

Higenamine protects neuronal cells from oxygen-glucose deprivation/reoxygenation-induced injury

Higenamine, a plant-based alkaloid, exhibits various properties, such as antiapoptotic and antioxidative effects. Previous studies proved that higenamine possesses potential therapeutic effects for ischemia/reperfusion (I/R) injuries. However, the role of higenamine in cerebral I/R injury has not been fully evaluated. Therefore, we aimed to investigate the effect of higenamine on cerebral I/R injury and the potential mechanism. Our data showed that higenamine ameliorated oxygen-glucose deprivation/reperfusion (OGD/R)-induced neuronal cells injury. Induction of reactive oxygen species and malonaldehyde production, and the inhibition of superoxide dismutase and glutathione peroxidase activity caused by OGD/R were attenuated by higenamine. In addition, higenamine inhibited the increases in caspase-3 activity and Bax expression, and inhibited the decrease in Bcl-2 expression. Furthermore, higenamine elevated the expression levels of p-Akt, heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2). The inhibitor of PI3K/Akt (LY294002) abolished the protective effects of higenamine on OGD/R-induced neuronal cells. These findings indicated that higenamine protects neuronal cells against OGD/R-induced injury by regulating the Akt and Nrf2/HO-1-signaling pathways. Collectively, higenamine might be considered as new strategy for the prevention and treatment of cerebral I/R injury.     

Ischemic post-conditioning protects brain and reduces inflammation in a rat model of focal cerebral ischemia/reperfusion

Ischemic post-conditioning (Post-cond) is a phenomenon in which intermittent interruptions of blood flow in the early phase of reperfusion can protect organ from ischemia/reperfusion (I/R) injury. Recent studies demonstrated ischemic Post-cond reduced infarct size in cerebral I/R injury. However, the molecular mechanisms underlying this phenomenon are not completely understood. As inflammation is known to be detrimental to the neurological outcome during the acute phase after stroke, we investigated whether ischemic Post-cond played its protective role in preventing post-ischemic inflammation in the rat middle cerebral artery occlusion model. Rats were treated with ischemic Post-cond after 60 min of occlusion (beginning of reperfusion). The infarct volume and myeloperoxidase activity were assessed at 24 h. The lipid peroxidation levels was evaluated by malondialdehyde assay and the expressions of interleukin-1β, tumor necrosis factor-α, and intercellular adhesion molecule 1 were studied by RT-PCR or western blotting. Ischemic Post-cond decreased myeloperoxidase activity and expressions of interleukin-1β, tumor necrosis factor-α, and intercellular adhesion molecule 1. Ischemic Post-cond also reduced infarct volume and lipid peroxidation levels. These findings indicated that ischemic Post-cond may be a promising neuroprotective approach for focal cerebral I/R injury and it is achieved, at least in part, by the inhibition of inflammation.     

The PGC-1α Activator ZLN005 Ameliorates Ischemia-Induced Neuronal Injury In Vitro and In Vivo

Oxidative stress is a great challenge to neurons following cerebral ischemia. PGC-1α has been shown to act as a potent modulator of oxidative metabolism. In this study, the effects of ZLN005, a small molecule that activate PGC-1α, against oxygen–glucose deprivation (OGD)- or ischemia-induced neuronal injury in vitro and in vivo were investigated. Transient middle cerebral artery occlusion (tMCAO) was performed in rats and ZLN005 was administered intravenously at 2 h, 4 h, or 6 h after ischemia onset. Infarct volume and neurological deficit score were detected to evaluate the neuroprotective effects of ZLN005. Well-differentiated PC12 cells, which were subjected to OGD for 2 h followed by reoxygenation for 22 h, were used as an in vitro ischemic model. Changes in expression of PGC-1α, its related genes, and antioxidant genes were determined by real-time quantitative PCR. The results showed that ZLN005 reduced cerebral infarct volume and improved the neurological deficit in rat with tMCAO, and significantly protected OGD-induced neuronal injury in PC12 cells. Furthermore, ZLN005 enhanced expression of PGC-1α in PC12 cells and in the ipsilateral hemisphere of rats with tMCAO. Additionally, ZLN005 increased antioxidant genes, including SOD1 and HO-1, and significantly prevented the ischemia-induced decrease in SOD activity. Taking together, the PGC-1α activator ZLN005 exhibits neuroprotective effects under ischemic conditions and molecular mechanisms possibly involve activation of PGC-1α signaling pathway and cellular antioxidant systems.     

Beneficial Effect of Astragalosides on Stroke Condition Using PC12 Cells under Oxygen Glucose Deprivation and Reperfusion

Astragalosides (AST) are reported to be neuroprotective in focal cerebral ischemic models in vivo. In this study, the direct effect of AST against oxygen and glucose deprivation (OGD) including neuronal injury and the underlying mechanisms in vitro were investigated. 5 h OGD followed by 24 h of reperfusion [adding back oxygen and glucose (OGD-R)] was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 µg/mL) were added to the culture after 5 h of the OGD ischemic insult and was present during the reoxygenation phases. A key finding was that OGD-R decreased cell viability, increased lactate dehydrogenase, increased reactive oxygen species, apoptosis, autophagy, functional impairment of mitochondria, and endoplasmic reticulum stress in PC12 cells, all of which AST treatment significantly reduced. In addition, AST attenuated OGD-R-induced cell loss through P38 MAPK activation a neuroprotective effect blunted by SB203580, a specific inhibitor of P38 MAPK. Our data suggest that both apoptosis and autophagy are important characteristics of OGD-R-induced PC12 death and that treating PC12 cells with AST blocked OGD-R-induced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and endoplasmic reticulum stress. Our data provide identification of AST that can concomitantly inhibit multiple cells death pathways following OGD injuries in neural cells.     

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.