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Ischemic stroke is a common disease that led to high mortality and high disability. NADPH oxidase 2- (NOX2-) mediated oxidative stress and long noncoding RNA have important roles in cerebral ischemia/reperfusion (CI/R) injury, whereas whether there is interplay between them remains to be clarified. This study was performed to observe the role of lncRNA PINK1-antisense RNA (PINK1-AS) in NOX2 expression regulation. An in vivo rat model (MCAO) and an in vitro cell model (H/R: hypoxia/reoxygenation) were utilized for CI/R oxidative stress injury investigation. The expression levels of lncRNA PINK1-AS, activating transcription factor 2 (ATF2), NOX2, and caspase-3 and the production level of ROS and cell apoptosis were significantly increased in CI/R injury model rats or in H/R-induced SH-SY5Y cells, but miR-203 was significantly downregulated. There was positive correlation between PINK1-AS expression level and ROS production level. PINK1-AS and ATF2 were found to be putative targets of miR-203. Knockdown of lncRNA PINK1-AS or ATF2 or the overexpression of miR-203 significantly reduced oxidative stress injury via inhibition of NOX2. Overexpression of lncRNA PINK1 significantly led to oxidative stress injury in SH-SY5Y cells through downregulating miR-203 and upregulating ATF2 and NOX2. lncRNA PINK1-AS and ATF2 were the targets of miR-203, and the lncRNA PINK1-AS/miR-203/ATF2/NOX2 axis plays pivotal roles in CI/R injury. Therefore, lncRNA PINK1-AS is a possible target for CR/I injury therapy by sponging miR-203., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Zhong-Bao Yang et al.)

NADPH oxidase 2 (NOX2) is a major subtype of NOX and is responsible for the generation of reactive oxygen species (ROS) in brain tissues. MicroRNAs (miRNAs/miRs) are important epigenetic regulators of NOX2. The present study aimed to identify the role of NOX2 miRNA‑targets in ischemic stroke (IS). A rat cerebral ischemia/reperfusion (CI/R) injury model and a SH‑SY5Y cell hypoxia/reoxygenation (H/R) model were used to simulate IS. Gene expression levels, ROS production and apoptosis in tissue or cells were determined, and bioinformatic analysis was conducted for target prediction of miRNA. In vitro experiments, including function‑gain and luciferase activity assays, were also performed to assess the roles of miRNAs. The results indicated that NOX2 was significantly increased in brain tissues subjected to I/R and in SH‑SY5Y cells subjected to H/R, while the expression of miR‑532‑3p (putative target of NOX2) was significantly decreased in brain tissues and plasma. Overexpression of miR‑532‑3p significantly suppressed NOX2 expression and ROS generation in SH‑SY5Y cells subjected to H/R, as well as reduced the relative luciferase activity of cells transfected with a reporter gene plasmid. Collectively, these data indicated that miR‑532‑3p may be a target of NOX2 and a biomarker for CI/R injury. Thus, the present study may provide a novel target for drug development and IS therapy.

Ischemic stroke is a devastating central nervous disease associated with oxidative stress and NOX2 is the main source of ROS responsible for brain tissue. miRNAs are a class of negative regulator of genes in mammals and involves the pathogenesis of ischemic stroke. This study aims to observe the role of target miRNA(miR-652) of NOX2 in ischemic stroke. A rat cerebral ischemia/reperfusion (CI/R) injury model and an SH-SY5Y cell hypoxia/reoxygenation(H/R) model were used to simulate ischemic stroke, and corresponding gene expression, biochemical indicators and pathophysiological indicators were measured to observe the role of miR-652. NOX2 significantly increased in brain tissues subjected to I/R or in SH-SY5Y cells subjected to H/R, while the expression level of miR-652(potential target of NOX2) significantly decreased in both brain tissues and plasma. Overexpression of miR-652 significantly suppressed NOX2 expression and ROS generation in H/R treated SH-SY5Y cells and reduced the relative luciferase activity of cells transfected with plasmid NOX2-WT (reporter gene plasmid). MiR-652 agomir significantly decreased the expression of NOX2 and ROS generation in brain tissues of CIR rats, as well as tissue injury. These data indicated that miR-652 protected rats from cerebral ischemia reperfusion injury by directly targeting NOX2, is a novel target for ischemic stroke therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background/aims: Ischemic stroke is still one of the leading debilitating diseases with high morbidity and mortality. NADPH oxidase (NOX)-derived reactive oxygen species (ROS) play an important role in cerebral ischemia/reperfusion (I/R) injury. However, the mechanism underlying the regulation of ROS generation is still not fully elucidated. This study aims to explore the role of transforming growth beta (TGF-β) signals in ROS generation., Methods: Sprague-Dawley rats were subjected to I/R injury, and PC-12 cells were challenged by hypoxia/reoxygenation (H/R) and/or treated with activin receptor-like kinase (ALK5) inhibitor Sb505124 or siRNA against ALK5. Brain damage was evaluated using neurological scoring, triphenyl tetrazolium chloride staining, hematoxylin and eosin staining, infarct volume measurement, TUNEL staining, and caspase-3 activity measurement. Expression of TGF-β and oxidative stress-related genes was analyzed by real-time polymerase chain reaction and Western blot; NOX activity and ROS level were measured using spectrophotometry and fluorescence microscopy, respectively., Results: I/R contributed to severe brain damage (impaired neurological function, brain infarction, tissue edema, apoptosis), TGF-β signaling activation (upregulation of ALK5, phosphorylation of SMAD2/3) and oxidative stress (upregulation of NOX2/4, rapid release of ROS [oxidative burst]). However, Sb505124 significantly reversed these alterations and protected rats against I/R injury. As in the animal results, H/R also contributed to TGF-β signaling activation and oxidative stress. Likewise, the inhibition of ALK5 or ALK5 knockdown significantly reversed these alterations in PC-12 cells. Other than ALK5 knockdown, ALK5 inhibition had no effect on the expression of ALK5 in PC-12 cells., Conclusions: Our studies demonstrated that TGF-β signaling activation is involved in the regulation of NOX2/NOX4 expression and exacerbates cerebral I/R injury., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

To explore the new mechanism of neuroprtection of monosialotetrahexosylganglioside and providing reliable theoretical foundation and experimental evidence for the emergency treatment and rehabilitation of cerebral ischemia/reperfusion injury. A rat model of cerebral ischemia/reperfusion injury was constructed and intervened with monosialotetrahexosylganglioside(5mg/kg) and lipid peroxidation inhibitor U-101033E(40mg/kg). TTC straining and neurobiological function score were used to evaluate brain injury. 4-HNE and MDA content were measured to evaluate lipid peroxidation. The expression of tyrosine hydroxilase at both mRNA and protein levels and enzyme activity were determined to evaluate the gene disfunction. Tyrosine content in brain and in serum and the DOPA content in plasma were measured to evaluate the metabolism of tyrosine. As the study shown, cerebral ischemia/reperfusion lead to brain infarction and neurobiological function losing accompany with upregulation of 4-HNE and MDA levels and downregulation of TH expression (mRNA and protein) and decreased enzyme activity. The results above mentioned can be reversed obviously by intervening with monosialotetrahexosylganglioside and lipid peroxidation inhibitor U-101033E. Toxic aldehyde accumulation leaded to disfunction of tyrosine hydroxylase and excessive tyrosine and decreased synthesis of catecholamine neurotransmitter such as dopamine and accelerated neuron cell injury. Both monosialotetrahexosylganglioside and U-101033E presented neuroprotecion by restoring the tyrosine/dopa pathway through reversing the function of tyrosine hydroxylase by inhibiting lipid peroxidation., (Copyright © 2016. Published by Elsevier Masson SAS.)

Objective Circulating microRNAs have been recognized as promising biomarkers for various diseases. The aim of the present study was to explore the potential role of circulating miR-107, miR-128b and miR-153 as non-invasive biomarkers in the diagnosis of ischemia stroke. Methods One hundred and fourteen ischemic stroke patients (61±11.3 years old) and 58 healthy volunteers (56±3.9 years old) matched for age and sex were enrolled in this study. Total RNA was isolated from plasma with TRIzol reagent. The circulating microRNAs levels were measured by quantitative real-time polymerase chain reaction. Results The circulating levels of miR-107, miR-128b and miR-153 significantly increased 2.78-, 2.13- and 1.83-fold in ischemia stroke patients in comparison to the healthy volunteers, respectively. Receiver operating characteristic (ROC) curves were analyzed using the SPSS software program and revealed the areas under the curve for circulating miR-107, miR-128b and miR-153 to be 0.97, 0.903 and 0.893 in ischemia stroke patients in comparison to healthy volunteers, respectively. The levels of circulating miR-107, miR-128b and miR-153 therefore positively correlated with the severity of stroke as defined by NIHSS classes. Conclusion Our results suggest that circulating miR-107, miR-128b and miR-153 might be used as potential novel non-invasive biomarkers for the diagnosis of ischemia stroke. However, future prospective trials in large-sized patient cohorts are needed before drawing any definitive conclusions.

Salviaolate is a group of depside salts isolated from Danshen (a traditional Chinese herbal medicine), with ≥ 85 % of magnesium lithospermate B. This study aims to investigate whether salviaolate is able to protect the rat brain from ischemia/reperfusion injury and the underlying mechanisms. Rats were subjected to 2 h of cerebral ischemia and 24 h of reperfusion to establish an ischemia/reperfusion injury model. The neuroprotective effects of salviaolate at different dosages were evaluated. A dosage (25 mg/kg) was chosen to explore the neuroprotective mechanisms of salviaolate. Neurological function, infarct volume, cellular apoptosis, nicotinamide adenine dinucleotide phosphate-oxidase activity, and H2O2 content were measured. In a nerve cell model of hypoxia/reoxygenation injury, magnesium lithospermate B was applied. Cellular apoptosis, lactate dehydrogenase, nicotinamide adenine dinucleotide phosphate-oxidase activity, and H2O2 content were examined. Ischemia/reperfusion treatment significantly increased the neurological deficit score, infarct volume, and cellular apoptosis accompanied by the elevated nicotinamide adenine dinucleotide phosphate-oxidase activity and H2O2 content in the rat brains. Administration of salviaolate reduced ischemia/reperfusion-induced cerebral injury in a dose-dependent manner concomitant with a decrease in nicotinamide adenine dinucleotide phosphate-oxidase activity and H2O2 production. Magnesium lithospermate B (20 mg/kg) and edaravone (6 mg/kg, the positive control) achieved the same beneficial effects as salviaolate did. In the cell experiments, the injury (indicated by apoptosis ratio and lactate dehydrogenase release), nicotinamide adenine dinucleotide phosphate-oxidase activity and H2O2 content were dramatically increased following hypoxia/reoxygenation, which were attenuated in the presence of magnesium lithospermate B (10(-5) M), VAS2870 (nicotinamide adenine dinucleotide phosphate-oxidase inhibitor), or edaravone (10(-5) M). The results suggest that salviaolate is able to protect the brain from ischemia/reperfusion oxidative injury, which is related to the inhibition of nicotinamide adenine dinucleotide phosphate-oxidase and a reduction of reactive oxygen species production., (Georg Thieme Verlag KG Stuttgart · New York.)

Recent studies have uncovered that accumulation of glutamate after ischaemic stroke is closely associated with the down-regulation of glutamate transporter-1 (GLT-1) expression, suggesting that GLT-1 expression critically controls glutamate accumulation and the abnormal glutamate transport-elicited neuronal cell excitotoxicity in patients with ischaemic stroke. However, it remains unknown how GLT-1 expression is regulated under ischaemic stroke conditions. In the present study, we screened the expression of nine brain-specific or brain-enriched miRNAs in a focal cerebral ischaemia/reperfusion (I/R) injury rat model, which showed glutamate accumulation and down-regulated GLT-1 expression as expected, and revealed that the miR-107 level was elevated in both brain tissue and plasma in the model. Next, we examined the functional relationship of miR-107 with GLT-1 expression in a nerve cell hypoxia/reoxygenation (H/R) injury model. H/R treatment increased apoptosis of the nerve cells concomitant with glutamate accumulation, miR-107 elevation and suppressed GLT-1 expression, mimicking our in vivo findings in the cerebral I/R injury rat model in vitro. Co-treating the cells with an miR-107 inhibitor blocked all of the effects, demonstrating that miR-107 functions to inhibit GLT-1 expression and elevate glutamate accumulation. To extend these animal and cell-based studies to clinical patients, we measured the plasma levels of miR-107 and glutamate, and observed that both miR-107 and glutamate were elevated in patients with ischaemic stroke. On the basis of these observations, we conclude that elevated miR-107 expression after ischaemic stroke accounts, at least partially, for glutamate accumulation through suppression of GLT-1 expression. Our findings also highlight that the plasma level of miR-107 may serve as a novel biomarker for monitoring excitotoxicity in patients with ischaemic stroke.

Many studies demonstrate that accumulation of reactive aldehydes plays an important role in cellular oxidative injury and aldehyde dehydrogenase 2 (ALDH2)-mediated detoxification of reactive aldehydes is thought as an endogenous protective mechanism against cell injury. This study was performed to explore whether Alda-1, a newly identified ALDH2 activator, was able to protect brain against ischemia/reperfusion injury through clearance of reactive aldehydes. In a rat model of focal cerebral ischemia/reperfusion injury, neurological function, infarct volume, cellular apoptosis, mortality, ALDH2 activity and protein expression, contents of 4-hydroxy-2-nonenal (4-HNE), and malondialdehyde (MDA) were determined. The results showed that ischemia/reperfusion treatment led to increase in neurological deficit score, infarct volume, cellular apoptosis, and mortality accompanied by the elevated levels of reactive aldehydes (4-HNE and MDA). There was no significant change in ALDH2 activity and protein expression. Alda-1 treatment at both dosages (15 mg/kg × 2 or 50 mg/kg × 2, i.g.) was able to increase the activity of ALDH2 and decrease the accumulation of reactive aldehydes concomitantly with the improvement of brain injury (decrease in infarct volume, cellular apoptosis, and mortality) and neurological function (decrease in neurological deficit score). However, Alda-1 treatment did not affect ALDH2 protein expression. Our results suggest that the protective effect of Alda-1 on cerebral ischemia/reperfusion injury is related to ALDH2 activation and clearance of reactive aldehydes.