ag-490 and Brain-Ischemia

Scutellarin is used to treat brain ischaemia. However, its underlying mechanism of action remains unclear. This study aimed to elucidate the potential mechanism of action of scutellarin in brain ischaemia through network pharmacology and experimental verification. The JAK2/STAT3 signalling pathway was identified and experimentally verified. Expression of JAK2/STAT3 signalling related proteins in TNC-1 astrocytes with BV-2 microglia-conditioned medium (CM), CM + lipopolysaccharide (LPS) (CM + L), and CM pretreated with scutellarin + LPS (CM + SL) was analysed by Western Blot and immunofluorescence staining. Expression levels of JAK2, p-JAK2, STAT3, and p-STAT3 were evaluated in astrocytes pre-treated with AG490. Middle cerebral artery occlusion (MCAO) in rats was performed in different experimental groups to detect expression of the above biomarkers. Network pharmacology suggested that the JAK2/STAT3 signalling pathway is one of the mechanisms by which scutellarin mitigates cerebral ischaemic damage. In TNC-1 astrocytes, p-JAK2 and p-STAT3 expression were significantly up-regulated in the CM + L group. Scutellarin promoted the up-regulation of various markers and AG490 neutralised the effect of scutellarin. In vivo, up-regulation of p-JAK2 and p-STAT3 after ischaemia is known. These results are consistent with previous reports. Scutellarin further enhanced this upregulation at 1, 3, and 7 d after MCAO. Scutellarin exerts its therapeutic effects on cerebral ischaemia by activating the astrocyte JAK2/STAT3 signalling, which provides a firm experimental basis for its clinical application.

Topics: Animals; Brain Injuries; Brain Ischemia; Culture Media, Conditioned; Janus Kinase 2; Lipopolysaccharides; Network Pharmacology; Rats

Cerebral ischemia/reperfusion injury is a severe problem in patients with brain ischemia. Brain injury caused by the immune response is important in the pathogenesis of cerebral ischemia/reperfusion injury and immune pathways. It is important to investigate potential targets for the treatment of cerebral ischemia/reperfusion injury.. In this experiment, we evaluated the effect of an exogenous JAK antagonist AG490 in the cerebral ischemia/reperfusion injury model, which was established by middle cerebral artery occlusion (MCAO). Histology study, TUNEL staining, Western blot, and RT-PCR were employed to examine the effects of AG490 in cerebral ischemia/reperfusion injury.. In the brain tissue of MCAO mice, JAK2 was highly expressed. AG490 is an inhibitor of JAK2, which reduced the phosphorylation level of JAK2. AG490 downregulated the phosphorylated activation of JAK3 and their downstream STAT3. The antiapoptotic activity of AG490 on cerebral ischemia/reperfusion injury mice was consistent with in vitro data. It reduced the phosphorylation of JAK2/JAK3/STAT3 and the apoptosis rate in cultured neurons upon apoptosis induction. Besides, we also observed the neuroprotective effects of AG490 on cerebral ischemia/reperfusion injury. Administration of AG490 could further enhance the expression of neurotrophins including BNDF, NT3, and the neurotrophin receptor TrkB.. Therefore, AG490 is pluripotent for cerebral ischemia/reperfusion injury through both antiapoptosis and neuroprotective activities. The antiapoptosis effect is dependent on its regulation of the JAK-STAT pathway.

Topics: Animals; Apoptosis; Brain Ischemia; Janus Kinase 2; Mice; Neuroprotective Agents; Reperfusion Injury; Signal Transduction; Tyrphostins

Previous studies have indicated that EPO maintains the M2 microglia phenotype that contributes to white matter repair after ischemic stroke in young mice (2 months old). However, the underlying mechanisms that regulate microglial polarization are poorly defined. This study investigated the neuroprotective effects of nonerythropoietic mutant EPO (MEPO) on white matter and the underlying mechanism in middle-aged (9-month-old) male mice following cerebral ischemia. Middle-aged male C57 BL/6 mice were treated with MEPO (5000 IU/kg) or vehicle after middle cerebral artery occlusion (MCAO) and reperfusion. The specific inhibitor AG490 was used to block the JAK2/STAT3 pathway. Neurological function was assessed by beam walking and adhesive removal tests. Immunofluorescence staining and western blotting were used to assess the severity of white matter injury, phenotypic changes in the microglia and the expression of the signaling molecules. MEPO significantly improved neurobehavioral outcomes, alleviated brain tissue loss, and ameliorated white matter injury after MCAO compared with the vehicle group. Moreover, MEPO promoted oligodendrogenesis by shifting microglia toward M2 polarization by promoting JAK2/STAT3 activation and inhibiting the expression of C/EBPβ at 14 days after cerebral ischemia-reperfusion. However, the MEPO's effect on microglial M2 polarization and oligodendrogenesis was largely suppressed by AG490 treatment. Collectively, these data indicate that MEPO treatment improves white matter integrity after cerebral ischemia, which may be partly explained by MEPO facilitating microglia toward the beneficial M2 phenotype to promote oligodendrogenesis via JAK2/STAT3 and the C/EBPβ signaling pathway. This study provides novel insight into MEPO treatment for ischemic stroke.

Topics: Animals; Behavior, Animal; Brain Ischemia; CCAAT-Enhancer-Binding Protein-beta; Cell Polarity; Erythropoietin; Janus Kinase 2; Male; Mice; Mice, Inbred C57BL; Microglia; Mutation; Oligodendroglia; Recovery of Function; Reperfusion Injury; Signal Transduction; STAT3 Transcription Factor; Steroid Isomerases; Tyrphostins; White Matter

This study aimed to investigate the effects of miR-1906 on cerebral ischemic injury and its underlying mechanisms. After 24 h of reperfusion, neurological deficit scores, brain water content and infarct volume were measured. Neuronal apoptosis was detected by using terminal dexynucleotidyl transferase-mediated dUTP nick end labeling assay. Hematoxylin-eosin staining was used to evaluate the histopathological damage of neurons. The expression of miR-1906 was detected by qRT-PCR. And the expressions of Bax, Bcl-2, caspase-3, Janus kinase 2 (JAK2), p-JAK2, signal transducer and activator of transcription 3 (STAT3) and p-STAT3 were measured by western blot. Furthermore, the levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and IL-6 were measured by ELISA. We found that miR-1906 expression was significantly decreased in the cerebral ischemia injury rats. miR-1906 decreased neurological score, infarct volume, brain water content, neuronal apoptosis and inflammatory factors (TNF-α, IL-6 and IL-1β) expression. In addition, miR-1906 promoted the phosphorylation of JAK2 and STAT3. After treating with JAK2/STAT3 pathway inhibitor AG490, the phosphorylation of JAK2 and STAT3 was inhibited and the effects of miR-1906 on neurological score, infarct volume, brain water content, neuronal apoptosis and inflammatory factors were reversed. miR-1906 could protect cerebral ischemic injury through activating the JAK2/STAT3 pathway in rats.

Topics: Animals; Apoptosis; Brain Ischemia; Janus Kinase 2; Male; MicroRNAs; Protective Agents; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; STAT3 Transcription Factor; Tyrphostins

Homocysteine (Hcy) has been shown to have a neurotoxic effect on ischemic brain cells; however, the underlying mechanisms remain incompletely understood. Here, we examined whether Hcy treatment influences mitochondria injury, oxidative stress, and mitochondrial STAT3 (mitoStat3) expression in rat ischemic brain. Our results demonstrated that Hcy treatment aggravated the damage of mitochondrial ultrastructure in the brain cortex and the dentate gyrus region of the hippocampus after focal cerebral ischemia. An elevated Hcy level was also accompanied by the significant inhibition of mitochondrial complex I-III enzymatic activities in addition to an increase in cytochrome c release. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) content and mitoStat3 protein phosphorylation level were increased in Hcy-treated animals, whereas AG490, a Jak2 inhibitor, inhibited mitoStat3 phosphorylation as well as 8-OHdG levels induced by Hcy. In vitro studies revealed that Hcy also markedly increased reactive oxygen species (ROS) and mitoStat3 levels. In addition, the inhibition of pSTAT3 reduced Hcy-mediated increase in ROS levels, whereas quenching ROS using the ROS inhibitor glutathione ethyl ester inhibited Hcy-mediated pSTAT3 overactivation in Neuro2a cells. These findings suggest that the development of therapies that interfere with the ROS/pSTAT3 pathway may be helpful for treating cerebral infarction-related diseases associated with Hcy.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Brain Ischemia; Cell Line; Cerebral Cortex; Dentate Gyrus; Deoxyguanosine; Gene Expression Regulation; Homocysteine; Male; Mitochondria; Mitochondrial Proteins; Oxidative Stress; Phosphorylation; Rats; Reactive Oxygen Species; STAT3 Transcription Factor; Tyrphostins

The purpose of this study was to investigate whether the kappa-opioid receptor (KOR) agonist, BRL52537, has a neuroprotective effect against cerebral ischemia/reperfusion (I/R) injury in rats and further explore the underlying mechanisms. Adult male Sprague-Dawley rats were randomly assigned into sham (group A), I/R (group B), BRL52537 (KOR agonist) + I/R (group C), nor-BNI (nor-binaltorphimine, KOR antagonist) + I/R (group D), AG490 (STAT3 phosphorylation inhibitor) + I/R (group E), dimethyl sulfoxide (DMSO, vehicle of AG490) + I/R (group F), and BRL52537 + AG490 +I/R (group G) groups. Cerebral I/R injury was induced by 10 min exposure to global ischemia (4-VO). Histopathological changes and neuronal apoptosis were evaluated with H&E staining and the TUNEL assay, respectively. Expression levels of signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3 and caspase-3 were determined with western blot analysis. Our results showed that BRL52537 protects against I/R injury-induced brain damage and inhibits neuronal apoptosis to a significant extent. Additionally, BRL52537 promoted up-regulation of p-STAT3 and a marked decrease in caspase-3 expression. Based on the collective findings, we propose that the KOR agonist, BRL52537, protects against cerebral I/R injury via a mechanism involving STAT3 signaling.

Topics: Animals; Apoptosis; Brain Ischemia; Caspase 3; Male; Neuroprotective Agents; Phosphorylation; Piperidines; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Reperfusion Injury; STAT3 Transcription Factor; Tyrphostins; Up-Regulation

Cerebral ischemia and reperfusion increase superoxide anions (O(2)(*-)) in brain mitochondria. Manganese superoxide dismutase (Mn-SOD; SOD2), a primary mitochondrial antioxidant enzyme, scavenges superoxide radicals and its overexpression provides neuroprotection. However, the regulatory mechanism of Mn-SOD expression during cerebral ischemia and reperfusion is still unclear. In this study, we identified the signal transducer and activator of transcription 3 (STAT3) as a transcription factor of the mouse Mn-SOD gene, and elucidated the mechanism of O(2)(*-) overproduction after transient focal cerebral ischemia (tFCI). We found that Mn-SOD expression is significantly reduced by reperfusion in the cerebral ischemic brain. We also found that activated STAT3 is usually recruited into the mouse Mn-SOD promoter and upregulates transcription of the mouse Mn-SOD gene in the normal brain. However, at early postreperfusion periods after tFCI, STAT3 was rapidly downregulated, and its recruitment into the Mn-SOD promoter was completely blocked. In addition, transcriptional activity of the mouse Mn-SOD gene was significantly reduced by STAT3 inhibition in primary cortical neurons. Moreover, we found that STAT3 deactivated by reperfusion induces accumulation of O(2)(*-) in mitochondria. The loss of STAT3 activity induced neuronal cell death by reducing Mn-SOD expression. Using SOD2-/+ heterozygous knock-out mice, we found that Mn-SOD is a direct target of STAT3 in reperfusion-induced neuronal cell death. Our study demonstrates that STAT3 is a novel transcription factor of the mouse Mn-SOD gene and plays a crucial role as a neuroprotectant in regulating levels of reactive oxygen species in the mouse brain.

Topics: Animals; Brain; Brain Infarction; Brain Ischemia; Cells, Cultured; Chromatin Immunoprecipitation; Cytochromes c; Disease Models, Animal; Electrophoretic Mobility Shift Assay; Embryo, Mammalian; Glucose; Humans; Hypoxia; Interleukin-6; Male; Mice; Mice, Knockout; Neurons; Neuroprotective Agents; Reperfusion; RNA, Small Interfering; STAT3 Transcription Factor; Superoxide Dismutase; Time Factors; Transfection; Tyrphostins; Up-Regulation

To investigate the neuroprotective effects and dose-response relation by combining JAK-STAT signal pathway inhibitor (AG490) with free radical scavenger dimethylthiourea (DMTU) in rats subjected to focal cerebral ischemia/reperfusion (I/R) injury.. In all rats, the middle cerebral artery occlusion (MCAO) was produced by occlusion of right internal carotid artery with a nylon monofilament. One hundred male Sprague-Dawley (SD) rats were divided into ten groups according to random digits table, 10 rats were in each group. The first experiment involved I/R model control, dimethyl sulfoxide (DMSO) control, normal saline (NS) control, AG490, DMTU and combination of AG490 and DMTU (A+D) groups. The second experiment involved model group and three experimental groups in which various doses of DMTU and AG490 were administered. The neurological behavior scores (NBS) were assessed at 24, 48 and 72 hours after reperfusion respectively in both experiments, and all the animals were then decapitated to determine the brain infarct volume after 72 hours.. The values of NBS in A+D group, AG490 group and DMTU group were higher than those in model group at 24, 48 and 72 hours after I/R, and their brain infarct volumes were obviously smaller than model group as well (all P<0.05). The brain infarct volume in A+D group was obviously smaller compared with AG490 and DMTU alone (all P<0.05). The values of NBS were higher and the brain infarct volumes were smaller in both high dose and medium dose combination groups than those in low dose combination and model groups respectively (all P<0.05). In addition, brain infarct volumes in high dose group were smaller than medium dose group (P<0.05), but there was no statistically significant difference between low dose and model groups.. The combined application of AG490 and DMTU produces a dose-dependent synergistic neuroprotective effect.

Topics: Animals; Brain; Brain Ischemia; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; Male; Neuroprotective Agents; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Thiourea; Tyrphostins