AZD1390, an ataxia telangiectasia mutated inhibitor, attenuates microglia-mediated neuroinflammation and ischemic brain injury.

AIMS: Excessive neuroinflammation mediated mainly by microglia plays a crucial role in ischemic stroke. AZD1390, an ataxia telangiectasia mutated (ATM) specific inhibitor, has been shown to promote radio-sensitization and survival in central nervous system malignancies, while the role of AZD1390 in ischemic stroke remains unknown. METHODS: Real-time PCR, western blot, immunofluorescence staining, flow cytometry and enzyme-linked immunosorbent assays were used to assess the activation of microglia and the release of inflammatory cytokines. Behavioral tests were performed to measure neurological deficits. 2,3,5-Triphenyltetrazolium chloride staining was conducted to assess the infarct volume. The activation of NF-κB signaling pathway was explored through immunofluorescence staining, western blot, co-immunoprecipitation and proximity ligation assay. RESULTS: The level of pro-inflammation cytokines and activation of NF-κB signaling pathway was suppressed by AZD1390 in vitro and in vivo. The behavior deficits and infarct size were partially restored with AZD1390 treatment in experimental stroke. AZD1390 restrict ubiquitylation and sumoylation of the essential regulatory subunit of NF-κB (NEMO) in an ATM-dependent and ATM-independent way respectively, which reduced the activation of the NF-κB pathway. CONCLUSION: AZD1390 suppressed NF-κB signaling pathway to alleviate ischemic brain injury in experimental stroke, and attenuated microglia activation and neuroinflammation, which indicated that AZD1390 might be an attractive agent for the treatment of ischemic stroke.

Inhibition of hippocampal cyclin-dependent kinase 5 activity ameliorates learning and memory dysfunction in a mouse model of bronchopulmonary dysplasia.

AIMS: Oxygen therapy plays a vital role in the development of bronchopulmonary dysplasia (BPD), which is the independent risk factor for neurodevelopment deficits in premature infants. However, the effect of hippocampal cyclin-dependent kinase 5 (CDK5) on BPD-associated neurodevelopment deficits is not fully understood. METHODS: Mice were placed in a hyperoxia chamber from postnatal Day 1 to Day 7. Hematoxylin and eosin staining was used to evaluate the lung histomorphological characteristics. Learning and memory functions of mice were detected by Morris water maze. TUNEL staining was applied to measure the number of apoptotic cells. The expression of CDK5, apoptosis-related protein, and neuroplasticity-related proteins were analyzed by Western blot. Golgi staining was used to assess the structure of dendritic spines. RESULTS: Hyperoxia-induced BPD mice showed a long-term learning and memory dysfunction, more severe neuronal apoptosis, and a decline of synaptic plasticity. Inhibition of CDK5 overactivation ameliorated cognitive deficits, neuronal apoptosis, and synaptic plasticity disorders in BPD mice. CONCLUSIONS: This study first found a vital role of CDK5 in BPD-associated neurodevelopmental disorders. Inhibition of CDK5 overexpression could effectively improve cognitive dysfunctions in BPD mice, which indicated that hippocampal CDK5 may be a new target for prevention and treatment in learning and memory dysfunction of BPD.

Fraxetin alleviates microglia-mediated neuroinflammation after ischemic stroke.

Background: Neuroinflammation, which is mainly mediated by excessive microglia activation, plays a major role in ischemic stroke. Overactivated microglia secrete numerous inflammatory cytokines, causing excessive inflammatory responses and ultimately exacerbating ischemic brain injury. Hence, compounds that attenuate neuroinflammation could become promising drug candidates for ischemic stroke. Fraxetin has an anti-inflammatory effect in many inflammatory diseases. However, whether it possesses an anti-inflammatory capacity in microglia-mediated neuroinflammation after ischemic brain injury is unknown. Our study aimed to investigate the suppression effect of fraxetin on neuroinflammation in lipopolysaccharide (LPS)-activated microglia and establish whether fraxetin could alleviate ischemic brain injury in a rodent model of ischemic stroke. Methods: For the in vitro experiment, primary microglia were obtained from 1-day-old C57/BL6J mice. The cells were activated with LPS and treated with fraxetin at a non-cytotoxic concentration. Real-time PCR, enzyme-linked immunosorbent assays, and immunofluorescence staining were used to evaluate the anti-inflammatory effects of fraxetin. The potential molecular mechanisms were explored and verified through RNA-sequencing analysis, western blotting and real-time PCR. For the in vivo experiment, focal ischemia was induced by middle cerebral artery occlusion (MCAO) in 8-week-old male C57/BL6J mice. Fraxetin (5 mg/kg) or an equal volume of saline was injected into mice intraperitoneally after MCAO, and 2% 2,3,5-triphenyltetrazolium chloride staining was applied to measure infarct volume. Behavioral tests were conducted to measure neurological deficits in the mice. Real-time PCR, western blotting, and immunofluorescence staining were used to examine the expression of inflammatory cytokines and microglia activation in the ischemic penumbra. Results: Fraxetin effectively inhibited the expression of proinflammatory cytokines including inducible nitric oxide synthase, tumor necrosis factor-α, interleukin-1 beta, and interleukin-6 in LPS-activated microglia. Fraxetin also suppressed the PI3K/Akt/NF-κB signaling pathway in activated microglia, which contributed to its anti-inflammatory effects. Furthermore, the administration of fraxetin attenuated ischemic brain injury and behavioral deficits after stroke. Finally, fraxetin was found to attenuate the activation of microglia both in vitro and in vivo. Conclusions: Our results suggest that fraxetin has a suppression effect on microglia-mediated neuroinflammation, and this effect is associated with the PI3K/Akt/NF-κB signaling pathway. Fraxetin may therefore have potential neuroprotective properties for ischemic stroke.

Imperatorin inhibits mitogen-activated protein kinase and nuclear factor kappa-B signaling pathways and alleviates neuroinflammation in ischemic stroke.

AIMS: Microglia-mediated neuroinflammation plays an important role in the pathological process of ischemic stroke, and the effect of imperatorin on post-stroke neuroinflammation is not fully understood. METHODS: Primary microglia were treated with imperatorin for 2 h followed by LPS (100 ng/ml) for 24 h. The expression of inflammatory cytokines was detected by RT-PCR, ELISA, and Western blot. The activation of MAPK and NF-κB signaling pathways were analyzed by Western blot. The ischemic insult was determined using a transient middle cerebral artery occlusion (tMCAO) model in C57BL/6J mice. Behavior tests were used to assess the neurological deficits of MCAO mice. TTC staining was applied to measure infract volume. RESULTS: Imperatorin suppressed LPS-induced activation of microglia and pro-inflammatory cytokines release and attenuated ischemic injury in MCAO mice. The results of transcriptome sequencing and Western blot revealed that downregulation of MAPK and NF-κB pathways might contribute to the protective effects of imperatorin. CONCLUSIONS: Imperatorin downregulated MAPK and NF-κB signaling pathways and exerted anti-inflammatory effects in ischemic stroke, which indicated that imperatorin might be a potential compound for the treatment of stroke.

LINC01198 promotes proliferation and temozolomide resistance in a NEDD4-1-dependent manner, repressing PTEN expression in glioma.

BACKGROUND: Dysregulation of numerous lncRNAs has been recently confirmed in glioma; however, the majority of their roles and mechanisms involved in this notorious disease remain largely unclear. This study aims to explore the roles and molecular mechanisms of LINC01198 implicated in the proliferation and chemoresistance in glioma. RESULTS: LINC01198 was elevated in glioma, and this predicted a poorer prognosis for patients with glioma. LINC01198 knockdown inhibited, while LINC01198 overexpression promoted, glioma cell proliferation and resistance to temozolomide. Mechanistically, NEDD4-1 (neural precursor cell expressed, developmentally downregulated 4, E3 ubiquitin protein ligase) and phosphatase and tensin homolog (PTEN) were recruited by LINC01198, which functioned as a scaffold. Moreover, we showed that LINC01198 exerted its oncogenic activities by enhancing the NEDD4-1-dependent repression of PTEN. CONCLUSIONS: Our study elucidated the role of oncogenic LINC01198 in glioma proliferation and temozolomide resistance, and this role may serve as a promising target for glioma therapy. METHODS: LINC01198 expression in glioma tissues and that in paired normal tissues were measured by qRT-PCR. The functional roles of LINC01198 in glioma were demonstrated by a series of in vitro experiments. CCK-8 assay, RNA pulldown, RNA immunoprecipitation and western blotting were used to demonstrate the potential mechanisms of LINC01198.

Effects of AQP5 gene silencing on proliferation, migration and apoptosis of human glioma cells through regulating EGFR/ERK/ p38 MAPK signaling pathway.

We investigated the effects of aquaporin 5 (AQP5) gene silencing on the proliferation, migration and apoptosis of human glioma cells through regulating the EGFR/ERK/p38MAPK signaling pathway. qRT-PCR was applied to examine the mRNA expressions of AQP5 in five human glioma cell lines. U87-MG, U251 and LN229 cells were selected and assigned into blank, vector, AQP5 siRNA and FlagAQP5 groups. MTT assay was used to measure cell proliferation. Flow cytometry (FCM) with AnnexinV-FITC/PI double staining and PI staining were employed to analyze cell apoptosis and cell cycle respectively. Scratch test was used to detect cell migration. Western blotting was performed to determine the EGFR/ERK/p38 MAPK signaling pathway-related proteins. Results showed that the positive expression of AQP5 in primary glioblastoma was associated with the tumor size and whether complete excision was performed. The mRNA expressions of AQP5 in cell lines of U87-MG, U251 and LN229 were significantly higher than in U373 and T98G. The proliferation rates of U87-MG, U251 and LN229 cells in the AQP5 siRNA group were lower than in the vector and blank groups. The apoptosis rate increased in the AQP5 siRNA group compared with the vector group. Scratch test demonstrated that AQP5 gene silencing could suppress cell migration. Compared with the vector and blank groups, the AQP5 siRNA group showed decreased expressions of the ERK1/2, p38 MAPK, p-ERK1/2 and p-p38 MAPK proteins. AQP5 gene silencing could inhibit the cell proliferation, reduce cell migration and promote the cell apoptosis of U87-MG, U251 and LN229 by suppressing EGFR/ERK/p38 MAPK signaling pathway.

IDH1R¹³²H decreases the proliferation of U87 glioma cells through upregulation of microRNA-128a.

Mutations in isocitrate dehydrogenase 1 (IDH1) are found in >70% of secondary glioblastomas and lower-grade gliomas (grades II-III). Among the numerous phenotypic differences between IDH1 mutant and wild-type glioma patients, the most salient is an improved survival rate for patients with a mutation. MicroRNAs (miRNAs) are a class of small, non­coding, single­stranded RNAs that can negatively regulate gene expression at the post­transcriptional level, predominantly by binding to the 3'­untranslated region of their target mRNAs. The dysregulated expression of several miRNAs has been reported to modulate glioma progression; however, it is unclear whether mutations in IDH1 regulate glioma cell proliferation through miRNA dysregulation. In the present study, stable overexpression of IDH1WT or IDH1R132H was established in the U87 glioma cell line. It was found that IDH1R132H decreased cell proliferation of U87 glioma cells by inducing the expression of the miRNA miR­128a. This process was dependent on the transcription factor hypoxia inducible factor­1α (HIF­1α), which binds to a hypoxia response element in the promoter of miR­128a. Furthermore, miR­128a negatively regulated the expression of B­cell­specific Moloney murine leukemia virus integration site 1 protein (Bmi­1), which is involved in suppressing cell proliferation. These findings suggest that the IDH1R132H­HIF­1α­miR­128a­Bmi­1 pathway is involved in glioma cell proliferation.

The up-regulation of voltage-gated sodium channel Nav1.6 expression following fluid percussion traumatic brain injury in rats.

BACKGROUND: The influx of Na and the depolarization mediated by voltage-gated sodium channels (VGSCs) is an early event in traumatic brain injury (TBI) induced cellular abnormalities and is therefore well positioned as an upstream target for pharmacologic modulation of the pathological responses to TBI. Alteration in the expression of the VGSC alpha-subunit has occurred in a variety of neuropathological states including focal cerebral ischemia, spinal injury, and epilepsy. OBJECTIVE: In this study, changes in Nav1.6 mRNA and protein expression were investigated in rat hippocampus after TBI. METHODS: Forty-eight adult male Sprague Dawley rats were randomly assigned to control or TBI groups. TBI was induced with a lateral fluid percussion device. Expression of mRNA and protein for Nav1.6 in the bilateral hippocampus was examined at 2, 12, 24, and 72 hours after injury by real-time quantitative polymerase chain reaction and Western blot. Immunofluorescence was performed to localize the expression of Nav1.6 protein in the hippocampus. RESULTS: Expression of >Nav1.6 mRNA was significantly up-regulated in the bilateral hippocampus at 2 and 12 hours post-TBI. Significant up-regulation of Nav1.6 protein was identified in the ipsilateral hippocampus from 2 to 72 hours post-TBI and in the contralateral hippocampus from 2 to 24 hours post-TBI. Expression of Nav1.6 occurred predominantly in neurons in the hippocampus. CONCLUSION: Results of the study showed significant up-regulation of mRNA and protein for Nav1.6 in rat hippocampal neurons after TBI.