Rongliang Wang

MiRNA-424 Protects Against Permanent Focal Cerebral Ischemia Injury in Mice Involving Suppressing Microglia Activation

Background and Purpose— We observed that microRNA-424 (miR-424) significantly decreased in an miRNA profile of circulating lymphocytes of patients with ischemic stroke. The present study focused on the potential and mechanism of miR-424 in protecting ischemic brain injury in mice. Methods— Cerebral ischemia was induced by middle cerebral artery occlusion in C57/BL6 mice. Cerebral infarction volume, neuronal apoptosis, and microglia activation were determined by 2,3,5-triphenyltetrazolium chloride staining, immunofluorescence, and Western blot. BV2 microglial cell activity, cell cycle, mRNA, and protein levels of miR-424 targets were accessed by enzyme-linked immunosorbent assay, flow cytometry, real-time polymerase chain reaction, and Western blot, respectively. Results— MiR-424 levels were decreased in the plasma of patients with acute ischemic stroke, as well as in mouse plasma and ipsilateral brain tissue at 4, 8, and 24 hours after ischemia, likewise, in the cortex, hippocampus, and basal ganglia, respectively, after 8-hour ischemia. Interestingly, pre- and post-treatment with overexpression of miR-424 both decreased cerebral infarction size and brain edema after middle cerebral artery occlusion. Meanwhile, lentiviral overexpression of miR-424 inhibited neuronal apoptosis and microglia activation, including suppressing ionized calcium binding adaptor molecule-1 immunoreactivity and protein level, and reduced tumor necrosis factor-&agr; production. In vitro study demonstrated that miR-424 mimics caused G1 phase cell-cycle arrest, inhibited BV2 microglia activity, and reduced the mRNA and protein levels of CDC25A, cyclin D1, and CDK6 in BV2 microglial cells, which were upregulated in brain of middle cerebral artery occlusion mice. Conclusions— MiR-424 overexpression lessened the ischemic brain injury through suppressing microglia activation by translational depression of key activators of G1/S transition, suggesting a novel miR-based intervention strategy for stroke.

MicroRNA-424 Protects Against Focal Cerebral Ischemia and Reperfusion Injury in Mice by Suppressing Oxidative Stress

Background and Purpose— We previously showed that the microRNA miR-424 protects against permanent cerebral ischemic injury in mice by suppressing microglia activation. This study investigated the role of miR-424 in transient cerebral ischemia in mice with a focus on oxidative stress–induced neuronal injury. Methods— Transient cerebral ischemia was induced in C57/BL6 mice by middle cerebral artery occlusion for 1 hour followed by reperfusion (ischemia/reperfusion). The miR-424 level in the peri-infarct cortex was quantified. Mice were also administered miR-424 angomir by intracerebroventricular injection. Cerebral infarct volume, neuronal apoptosis, and levels of oxidative stress markers and antioxidants were evaluated. In an in vitro experiment, primary cortical neurons were exposed to H2O2 and treated with miR-424 angomir, nuclear factor erythroid 2-related factor 2 siRNA, and superoxide dismutase (SOD) inhibitor; cell activity, lactate dehydrogenase release, malondialdehyde level, and manganese (Mn)SOD activity were then evaluated. Results— MiR-424 levels in the peri-infarct cortex increased at 1 and 4 hours then decreased 24 hours after reperfusion. Treatment with miR-424 decreased infarct volume and inhibited neuronal apoptosis after ischemia/reperfusion, reduced reactive oxygen species and malondialdehyde levels in the cortex, and increased the expression and activation of MnSOD as well as the expression of extracellular SOD and the redox-sensitive transcription factor nuclear factor erythroid 2-related factor. In neuronal cultures, miR-424 treatment abrogated H2O2-induced injury, as evidenced by decreased lactate dehydrogenase leakage and malondialdehyde level and increased cell viability and MnSOD activity; the protective effects of miR-424 against oxidative stress were reversed by nuclear factor erythroid 2-related factor knockdown and SOD inhibitor treatment. Conclusions— MiR-424 protects against transient cerebral ischemia/reperfusion injury by inhibiting oxidative stress.

MicroRNA-23a-3p attenuates oxidative stress injury in a mouse model of focal cerebral ischemia-reperfusion.

The present study was designed to investigate the potential role of miR-23a-3p in experimental brain ischemia-reperfusion injury. Cerebral ischemia reperfusion was induced by transient middle cerebral artery occlusion (MCAO) for 1h in C57/BL6 mice. And miR-23a-3p angomir was transfected to upregulate the miR-23a-3p level. Our results showed that miR-23a-3p levels were transiently increased at 4h after reperfusion in the peri-infarction area, while markedly increased in the infarction core at reperfusion 4h and 24h. Importantly, in vivo study demonstrated that miR-23a-3p angomir treatment through intracerebroventricular injection markedly decreased cerebral infarction volume after MCAO. Simultaneously, miR-23a-3p reduced peroxidative production nitric oxide (NO) and 3-nitrotyrosine (3-NT), and increased the expression of manganese superoxide dismutase (MnSOD). In vitro study demonstrated that miR-23a-3p decreased hydrogen peroxide (H2O2)-induced lactate dehydrogenase (LDH) leakage dose-dependently, and reduced protein levels of activated caspase-3 in neuro-2a cells. In addition, miR-23a-3p reduced H2O2-induced production of NO and 3-NT dose-dependently, and reversed the decreased activity of total SOD and MnSOD in neuro-2a cells. Our study indicated that miR-23a-3p suppressed oxidative stress and lessened cerebral ischemia-reperfusion injury.

AKT/GSK3β-dependent autophagy contributes to the neuroprotection of limb remote ischemic postconditioning in the transient cerebral ischemic rat model.

Limb remote ischemic postconditioning (RIPostC) has been recognized as an applicable strategy in protecting against cerebral ischemic injury. However, the time window for application of limb RIPostC and the mechanisms behind RIPostC are still unclear.

Neuroprotective effect of microRNA-99a against focal cerebral ischemia-reperfusion injury in mice.

MicroRNA-99a (miR-99a) has been reported to function as a tumor suppressor through regulating cell cycle and apoptosis. But its clinical significance in ischemic stroke and its function in cerebral ischemia-reperfusion (I/R) injury remained unknown. Herein transient middle cerebral artery occlusion was built on C57BL/6 mice, followed by intracerebroventricular injection of miR-99a agomir or antagomir before reperfusion for 24h. Our clinical analysis indicates that plasma miR-99a level was significantly decreased in ischemic stroke patients as compared to healthy subjects, and a significant correlation was observed between miR-99a and clinical parameters. And miR-99a overexpression mitigated I/R injury in mice, as evidenced by reduced brain infarct volume and neural apoptosis, whereas miR-99a downregulation aggravates brain injury. In vitro, miR-99a protected neuro-2a cells against hydrogen peroxide-induced oxidative stress injury, by improving cell viability, suppressing LDH release and cell apoptosis. In addition, miR-99a overexpression inhibited H2O2 induced G1/S phase transition in neuro-2a cells, accompanied by a significant decrease in cyclin D1 level and a tendency of down-regulation of CDK6. It was further proved in mice that miR-99a inhibited cyclin D1 and CDK6 expressions following cerebral I/R injury. These findings indicate that miR-99a reduces neuronal damage following cerebral I/R through regulating cell cycle progression and preventing apoptosis, suggesting that miR-99a could be used as a new therapeutic agent targeting neuronal cell cycle re-entry following stroke.

MicroRNA-181c Exacerbates Brain Injury in Acute Ischemic Stroke

MicroRNA-181 (miR-181) is highly expressed in the brain, and downregulated in miRNA expression profiles of acute ischemic stroke patients. However, the roles of miR-181c in stroke are not known. The clinical relevance of miR-181c in acute stroke patients was evaluated by real-time PCR and correlation analyses. Proliferation and apoptosis of BV2 microglial cells and Neuro-2a cells cultured separately or together under oxidative stress or inflammation were assessed with the Cell Counting Kit-8 and by flow cytometry, respectively. Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in C57/BL6 mice, and cerebral infarct volume, microglia activation, and expression of pro-apoptotic factors were evaluated by 2,3,5-triphenyl-2H-tetrazolium chloride staining, immunocytochemistry, and western blotting, respectively. Plasma levels of miR-181c were decreased in stroke patients relative to healthy individuals, and were positively correlated with neutrophil number and blood platelet count and negatively correlated with lymphocyte number. Lipopolysaccharide (LPS)/hydrogen peroxide (H2O2) treatment inhibited BV2 microglia proliferation without inducing apoptosis, while miR-181c reduced proliferation but increased the apoptosis of these cells with or without LPS/H2O2 treatment. LPS/H2O2 induced apoptosis in Neuro-2a cells co-cultured with BV2 cells, an effect that was potentiated by miR-181c. In the MCAO model, miR-181c agomir modestly increased infarct volume, markedly decreased microglia activation and B cell lymphoma-2 expression, and increased the levels of pro-apoptotic proteins in the ischemic brain. Our data indicate that miR-181c contributes to brain injury in acute ischemic stroke by promoting apoptosis of microglia and neurons via modulation of pro- and anti-apoptotic proteins.

Ischemic Postconditioning Relieves Cerebral Ischemia and Reperfusion Injury Through Activating T-LAK Cell–Originated Protein Kinase/Protein Kinase B Pathway in Rats

Background and Purpose— Ischemic postconditioning (IPostC) protects against ischemic brain injury. To date, no study has examined the role of T-LAK-cell–originated protein kinase (TOPK) in IPostC-afforded neuroprotection. We explored the molecular mechanism related with TOPK in antioxidant effect of IPostC against ischemia/reperfusion. Methods— Focal ischemia was induced in rats by transient middle cerebral artery occlusion. Reactive oxygen species production in the peri-infarct cortex was detected using dihydroethidium. Malondialdehyde, as a marker of lipid peroxidation, and 3-nitrotyrosine, as a marker of protein oxidation, were detected by ELISA. The expression or location of antioxidant proteins and signal molecules TOPK, phosphatase, and tensin homolog, and Akt was analyzed by Western blotting and immunofluorescence. Results— Our results revealed that IPostC relieved transient middle cerebral artery occlusion–induced oxidative damage by reducing reactive oxygen species, malondialdehyde, and 3-nitrotyrosine accumulation in the peri-infarct cortex and raised levels of antioxidants perioxiredoxin-1, peroxiredoxin-2, and thioredoxin-1. In addition, IPostC increased p-AKT and p-TOPK levels, which colocalized in neural cells. In vitro TOPK knockdown by small interfering RNA decreased the levels of antioxidants peroxiredoxin-1, thioredoxin, and manganese superoxide dismutase activity in PC12 cells. In vivo intracerebroventricular injection of TOPK small interfering RNA reversed IPostC-induced neuroprotection by increasing infarct volume and nitric oxide content and reducing manganese superoxide dismutase activity. Moreover, IPostC-evoked Akt activation was blocked by TOPK small interfering RNA in vivo, but the decreased phosphorylated phosphatase and tensin homolog level in ischemia/reperfusion was not influenced by IPostC or by TOPK small interfering RNA treatment. Conclusions— Our results suggest that the antioxidative effects of TOPK/Akt might contribute to the neuroprotection of IPostC treatment against transient middle cerebral artery occlusion.

Long Noncoding RNA H19 Promotes Neuroinflammation in Ischemic Stroke by Driving Histone Deacetylase 1–Dependent M1 Microglial Polarization

Background and Purpose— Long noncoding RNA H19 is repressed after birth, but can be induced by hypoxia. We aim to investigate the impact on and underlying mechanism of H19 induction after ischemic stroke. Methods— Circulating H19 levels in stroke patients and mice subjected to middle cerebral artery occlusion were assessed using real-time polymerase chain reaction. H19 siRNA and histone deacetylase 1 (HDAC1) plasmid were used to knock down H19 and overexpress HDAC1, respectively. Microglial polarization and ischemic outcomes were assessed in middle cerebral artery occlusion mice and BV2 microglial cells subjected to oxygen–glucose deprivation. Results— Circulating H19 levels were significantly higher in stroke patients compared with healthy controls, indicating high diagnostic sensitivity and specificity. Moreover, plasma H19 levels showed a positive correlation with National Institute of Health Stroke Scale score and tumor necrosis factor-&agr; levels. After middle cerebral artery occlusion in mice, H19 levels increased in plasma, white blood cells, and brain. Intracerebroventricular injection of H19 siRNA reduced infarct volume and brain edema, decreased tumor necrosis factor-&agr; and interleukin-1&bgr; levels in brain tissue and plasma, and increased plasma interleukin-10 concentrations 24 hours poststroke. Additionally, H19 knockdown attenuated brain tissue loss and neurological deficits 14 days poststroke. BV2 cell-based experiments showed that H19 knockdown blocked oxygen–glucose deprivation–driven M1 microglial polarization, decreased production of tumor necrosis factor-&agr; and CD11b, and increased the expression of Arg-1 and CD206. Furthermore, H19 knockdown reversed oxygen–glucose deprivation–induced upregulation of HDAC1 and downregulation of acetyl-histone H3 and acetyl-histone H4. In contrast, HDAC1 overexpression negated the effects of H19 knockdown. Conclusions— Our findings indicate that H19 promotes neuroinflammation by driving HDAC1-dependent M1 microglial polarization, suggesting a novel H19-based diagnosis and therapy for ischemic stroke.

Improvement of hematoma absorption and neurological function in patients with acute intracerebral hemorrhage treated with Xueshuantong

Spontaneous intracerebral hemorrhage (ICH) leads to high mortality and morbidity. Currently, there is no effective therapy for ICH. Herein we conducted a clinical study in patients with acute ICH to investigate the efficacy of Xueshuantong Injection, a Chinese herbal prescription known for treatment of ischemic diseases in China. Patients (n=63) were randomly assigned to control (n=29) and Xueshuantong Injection treatment (175 mg/d, n=34) groups. Both groups were evaluated using their history and vital signs. The National Institutes of Health Stroke Scale (NIHSS) scores, hematoma volume by CT scanning, and inflammatory factors were assessed before and after two weeks treatment. There were no significant differences in all parameters between two groups before treatment. The treatment group showed significant decreases in both NIHSS score and hematoma volume, compared to control group after treatment (P<0.01 and P<0.05, respectively). Furthermore, the inflammatory factors, as measured by leukocytes, neutrophil percentage and C-reactive protein values, were significantly reduced in treatment group compared to control group after treatment (P<0.05, P<0.05, P<0.01 respectively). Our results showed that treatment with Xueshuantong Injection reduced inflammatory response and increased hematoma absorption, which significantly improved recovery of neurological function. This suggests Xueshuantong Injection as a potential treatment of patients with acute ICH.

Intra-artery infusion of recombinant human erythropoietin reduces blood-brain barrier disruption in rats following cerebral ischemia and reperfusion

Objectives: Intra-artery infusion of recombinant human erythropoietin (rhEPO) has recently been reported to confer neuroprotection against cerebral ischemia-reperfusion injury in animal models; however, the molecular mechanisms are still under investigation. The present study focused on the specific mechanism involved in blood–brain barrier (BBB) disruption. Methods: Thirty-six male and nine female Sprague Dawley rats were subjected to middle cerebral artery (MCA) occlusion to induce focal cerebral ischemia, and administrated rhEPO at a dose of 800 U/kg through MCA infusion at the beginning of reperfusion. Neurobehavioral deficits, brain edema, and infarct volume were evaluated after 2 h of ischemia and 24 h of reperfusion. BBB permeability was assessed by quantifying the extravasation of Evans blue (EB) dye. The expression of tight junction proteins and matrix metalloproteinases (MMPs) (Claudin-5, Occludin, MMP-2, and MMP-9) in microvessels were detected by immunofluorescence and western blot. The activities of MMPs in the cerebral microvessels were determined by gelatin zymography. Results: Treatment with rhEPO through the MCA strongly alleviated infarct volume, brain edema, and improved neurobehavioral outcomes in male and female rats. In addition, rhEPO remarkably suppressed the EB extravasation induced by brain ischemia. Furthermore, rhEPO prevented degradation of Claudin-5 and Occludin, and reduced the expression and activity of MMP-2 and MMP-9 in isolated brain microvessels. Conclusions: Treatment with rhEPO through MCA infusion prevented brain edema formation and infarction through inhibition of MMP-mediated BBB disruption in acute ischemic stroke.