Effect of Celastrol on LncRNAs and mRNAs Profiles of Cerebral Ischemia-Reperfusion Injury in Transient Middle Cerebral Artery Occlusion Mice Model
ABSTRACT: Celastrol plays a significant role in cerebral ischemia-reperfusion injury. Although previous studies have confirmed that celastrol post-treatment has a protective effect on ischemic stroke, the therapeutic effect of celastrol on ischemic stroke and the underlying molecular mechanism remain unclear. In the present study, focal transient cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in mice and celastrol was administered immediately after reperfusion. We performed lncRNA and mRNA analysis in the ischemic hemisphere of adult mice with celastrol post-treatment through RNA-Sequencing (RNA-Seq). A total of 50 differentially expressed lncRNAs (DE lncRNAs) and 696 differentially expressed mRNAs (DE mRNAs) were identified between the sham and tMCAO group, and a total of 544 DE lncRNAs and 324 DE mRNAs were identified between the tMCAO and tMCAO+celastrol group. Bioinformatic analysis was done on the identified deregulated genes through gene ontology (GO) analysis, KEGG pathway analysis and network analysis. Pathway analysis indicated that inflammation-related signaling pathways played vital roles in the treatment of ischemic stroke by celastrol. Our study suggests celastrol treatment can effectively reduce cerebral ischemia-reperfusion injury. The bioinformatics analysis of lnRNAs and mRNAs profiles in the ischemic hemisphere of adult mice provides a new perspective in the neuroprotective effects of celastrol, particularly with regards to ischemic stroke.
Project description:Celastrol, a pentacyclic triterpene isolated from the traditional Chinese medicine Tripterygium wilfordii Hook. F., exhibits effectiveness in protection against multiple central nervous system (CNS) diseases such as cerebral ischemia, but its influence on lipidomics still remains unclear. Therefore, in the present study, the efficacy and potential mechanism of celastrol against cerebral ischemia/reperfusion (I/R) injury were investigated based on lipidomics. Middle cerebral artery occlusion (MCAO) followed by reperfusion was operated in mice to set up a cerebral I/R model. TTC staining and TUNEL staining were used to evaluate the therapeutic effect of celastrol. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS) was employed for lipidomics analysis in ipsilateral hemisphere and plasma. Celastrol remarkably reduced cerebral infarct volume and apoptosis positive cells in tMCAO mice. Furthermore, lipidomics analysis showed that 14 common differentially expressed lipids (DELs) were identified in brain and five common DELs were identified in plasma between the Sham, tMCAO and Celastrol-treated tMCAO groups. Through enrichment analysis, sphingolipid metabolism and glycerophospholipid metabolism were demonstrated to be significantly enriched in all the comparison groups. Among the DELs, celastrol could reverse cerebral I/R injury-induced alteration of phosphatidylcholine, phosphatidylethanolamine and sulfatide, which may be responsible for the neuroprotective effect of celastrol. Our findings suggested the neuroprotection of celastrol on cerebral I/R injury may be partially associated with its regulation of lipid metabolism.
Project description:BACKGROUND:The transient middle cerebral artery occlusion (tMCAO) model is used for studying the molecular mechanisms of ischemic damage and neuroprotection. Numerous studies have demonstrated the role of individual genes and associated signaling pathways in the pathogenesis of ischemic stroke. Here, the tMCAO model was used to investigate the genome-wide response of the transcriptome of rat brain tissues to the damaging effect of ischemia and subsequent reperfusion. RESULTS:Magnetic resonance imaging and histological examination showed that the model of focal ischemia based on endovascular occlusion of the right middle cerebral artery for 90 min using a monofilament, followed by restoration of the blood flow, led to reproducible localization of ischemic damage in the subcortical structures of the brain. High-throughput RNA sequencing (RNA-Seq) revealed the presence of differentially expressed genes (DEGs) in subcortical structures of rat brains resulting from hemisphere damage by ischemia after tMCAO, as well as in the corresponding parts of the brains of sham-operated animals. Real-time reverse transcription polymerase chain reaction expression analysis of 20 genes confirmed the RNA-Seq results. We identified 469 and 1939 genes that exhibited changes in expression of >?1.5-fold at 4.5 and 24 h after tMCAO, respectively. Interestingly, we found 2741 and 752 DEGs under ischemia-reperfusion and sham-operation conditions at 24 h vs. 4.5 h after tMCAO, respectively. The activation of a large number of genes involved in inflammatory, immune and stress responses, apoptosis, ribosome function, DNA replication and other processes was observed in ischemia-reperfusion conditions. Simultaneously, massive down-regulation of the mRNA levels of genes involved in the functioning of neurotransmitter systems was recorded. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that dozens of signaling pathways were associated with DEGs in ischemia-reperfusion conditions. CONCLUSIONS:The data obtained revealed a global profile of gene expression in the rat brain sub-cortex under tMCAO conditions that can be used to identify potential therapeutic targets in the development of new strategies for the prevention and treatment of ischemic stroke.
Project description:<h4>Background</h4>Converging evidence suggests that inflammatory processes significantly influence brain injury and clinical impairment in ischemic stroke. Although early studies suggested a key role of lymphocytes, recent data has emphasized the orchestrating function of innate immunity, i.e., macrophages and microglia. The bifunctional receptor and ectoenzyme CD38 synthesizes calcium-mobilizing second messengers (e.g., cyclic ADP-ribose), which have been shown to be necessary for activation and migration of myeloid immune cells. Therefore, we investigated the dynamics of CD38 in stroke and the impact of CD38-deficiency on cytokine production, inflammation and cerebral damage in a mouse model of cerebral ischemia-reperfusion.<h4>Methodology/principal findings</h4>We show that the local expression of the chemokine MCP-1 was attenuated in CD38-deficient mice compared with wildtype mice after focal cerebral ischemia and reperfusion. In contrast, no significant induction of MCP-1 expression was observed in peripheral blood after 6 hours. Flow cytometry analysis revealed less infiltrating macrophages and lymphocytes in the ischemic hemisphere of CD38-deficient mice, whereas the amount of resident microglia was unaltered. An up-regulation of CD38 expression was observed in macrophages and CD8(+) cells after focal cerebral ischemia in wildtype mice, whereas CD38 expression was unchanged in microglia. Finally, we demonstrate that CD38-deficiency decreases the cerebral ischemic injury and the persistent neurological deficit after three days of reperfusion in this murine temporary middle cerebral artery occlusion (tMCAO) model.<h4>Conclusion/significance</h4>CD38 is differentially regulated following stroke and its deficiency attenuates the postischemic chemokine production, the immune cell infiltration and the cerebral injury after temporary ischemia and reperfusion. Therefore CD38 might prove a therapeutic target in ischemic stroke.
Project description:This study aimed to explore the differential expression profiles of lncRNAs of brain between the sham group and cerebral ischemia mouse. Methods: In the present study, focal transient cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in mice. LncRNA profiles of brain tissues in IS and controls were generated by high-throughput sequencing. The sequence reads that passed quality filters were analyzed at the transcript isoform level with an Illumina HiSeqTM 2500. qRT-PCR validation was performed using SYBR Green assays. Results: The results showed that between the sham group and I/R group, 249 lncRNAs were upregulated and 5 lncRNAs were downregulated (fold change≥2, P < 0.05, treat >1). Bioinformatic analysis was done on the identified deregulated genes through gene ontology (GO) analysis, KEGG pathway analysis and network analysis. The bioinformatics analysis of lncRNAs and mRNAs profiles in the ischemic hemisphere of adult mice provides a new perspective in the neuroprotective effects of celastrol, particularly with regards to ischemic stroke.
Project description:Hyperbaric oxygen (HBO) and normobaric hyperoxia (NBO) protect the brain parenchyma and the cerebral microcirculation against ischemia. We studied their effect on secondary hemorrhage after thrombolysis in two thromboembolic middle cerebral artery occlusion (MCAO) (tMCAO) models. Beginning 60 minutes after tMCAO with either thrombin-induced thromboemboli (TT) or calcium-induced thromboemboli (CT), spontaneously hypertensive rats (n=96) breathed either air, 100% O(2) (NBO), or 100% O(2) at 3 bar (HBO) for 1 hour. Immediately thereafter, recombinant tissue plasminogen activator (rt-PA, 9 mg/kg) was injected. Although significant reperfusion was observed after thrombolysis in TT-tMCAO, vascular occlusion persisted in CT-tMCAO. In TT-tMCAO, NBO and HBO significantly reduced diffusion-weighted imaging-magnetic resonance imaging (MRI) lesion volume and postischemic blood-brain barrier (BBB) permeability on postcontrast T1-weighted images. NBO and, significantly more potently, HBO reduced macroscopic hemorrhage on T2* MRI and on corresponding postmortem cryosections. Oxygen therapy lowered hemoglobin content and attenuated activation of matrix metalloproteinases in the ischemic hemisphere. In contrast, NBO and HBO failed to reduce infarct size in CT but both decreased BBB damage and microscopic hemorrhagic transformation. Only HBO reduced hemoglobin extravasation in the ischemic hemisphere. In conclusion, NBO and HBO decrease infarct size after thromboembolic ischemia only if recanalization is successful. As NBO and HBO also reduce postthrombolytic intracerebral hemorrhage, combining the two with thrombolysis seems promising.
Project description:Ischemic brain stroke is one of the most serious and socially significant diseases. In addition to messenger RNAs (mRNAs), encoding protein, the study of regulatory RNAs in ischemic has exceptional importance for the development of new strategies for neuroprotection. Circular RNAs (circRNAs) have a closed structure, predominantly brain-specific expression, and remain highly promising targets of research. They can interact with microRNAs (miRNAs), diminish their activity and thereby inhibit miRNA-mediated repression of mRNA. Genome-wide RNA-Seq analysis of the subcortical structures of the rat brain containing an ischemic damage focus and penumbra area revealed 395 circRNAs changed their expression significantly at 24 h after transient middle cerebral artery occlusion model (tMCAO) conditions. Furthermore, functional annotation revealed their association with neuroactive signaling pathways. It was found that about a third of the differentially expressed circRNAs (DECs) originate from genes whose mRNA levels also changed at 24 h after tMCAO. The other DECs originate from genes encoding non-regulated mRNAs under tMCAO conditions. In addition, bioinformatic analysis predicted a circRNA-miRNA-mRNA network which was associated with the neurotransmission signaling regulation. Our results show that such circRNAs can persist as potential miRNA sponges for the protection of mRNAs of neurotransmitter genes. The results expanded our views about the neurotransmission regulation in the rat brain after ischemia-reperfusion with circRNA action.
Project description:<h4>Background</h4>Brain ischemia is known to include neuronal cell death and persisting neurological deficits. A lack of oxygen and glucose are considered to be key mediators of ischemic neurodegeneration while the exact mechanisms are yet unclear. In former studies the expression of two different two-pore domain potassium (K2P) channels (TASK1, TREK1) were shown to ameliorate neuronal damage due to cerebral ischemia. In neurons, TASK channels carrying hyperpolarizing K+ leak currents, and the pacemaker channel HCN2, carrying depolarizing Ih, stabilize the membrane potential by a mutual functional interaction. It is assumed that this ionic interplay between TASK and HCN2 channels enhances the resistance of neurons to insults accompanied by extracellular pH shifts.<h4>Methods</h4>In C57Bl/6 (wildtype, WT), hcn2+/+ and hcn2-/- mice we used an in vivo model of cerebral ischemia (transient middle cerebral artery occlusion (tMCAO)) to depict a functional impact of HCN2 in stroke formation. Subsequent analyses comprise behavioural tests and hcn2 gene expression assays.<h4>Results</h4>After 60 min of tMCAO induction in WT mice, we collected tissue samples at 6, 12, and 24 h after reperfusion. In the infarcted neocortex, hcn2 expression analyses revealed a nominal peak of hcn2 expression 6 h after reperfusion with a tendency towards lower expression levels with longer reperfusion times. Hcn2 gene expression levels in infarcted basal ganglia did not change after 6 h and 12 h. Only at 24 h after reperfusion, hcn2 expression significantly decreases by ~55%. However, 30 min of tMCAO in hcn2-/- as well as hcn2+/+ littermates induced similar infarct volumes. Behavioural tests for global neurological function (Bederson score) and motor function/coordination (grip test) were performed at day 1 after surgery. Again, we found no differences between the groups.<h4>Conclusions</h4>Here, we hypothesized that the absence of HCN2, an important functional counter player of TASK channels, affects neuronal survival during stroke-induced tissue damage. However, together with a former study on TASK3 these results implicate that both TASK3 and HCN2 which were supposed to be neuroprotective due to their pH-dependency, do not influence ischemic neurodegeneration during stroke in the tMCAO model.
Project description:BACKGROUND:Ischemic stroke-induced neuroinflammation is mainly mediated by microglial cells. The nuclear factor kappa B (NF-?B) pathway is the key transcriptional pathway that initiates inflammatory responses following cerebral ischemia. OTULIN, a critical negative regulator of the NF-?? signaling pathway, exerts robust effects on peripheral immune cell-mediated inflammation and is regarded as an essential mediator for repressing inflammation in vivo. The effect of OTULIN on inflammatory responses in the central nervous system (CNS) was previously unstudied. This current study investigated the anti-inflammatory effect of OTULIN both in vitro and in vivo in ischemic stroke models. METHODS:Sprague-Dawley (SD) rats were subjected to transient middle cerebral artery occlusion (tMCAO) or an intraperitoneal injection of lipopolysaccharide (LPS). Overexpression of the OTULIN gene was utilized to observe the effect of OTULIN on ischemic stroke outcomes. The effect of OTULIN overexpression on microglia-mediated neuroinflammation was examined in rat primary microglia (PM) and in the microglial cell line N9 after induction by oxygen-glucose deprivation (OGD)-treated neuronal medium. The activation and inflammatory responses of microglia were detected using immunofluorescence, ELISA, and qRT-PCR. The details of molecular mechanism were assessed using Western blotting. RESULTS:In the tMCAO rats, the focal cerebral ischemia/reperfusion injury induced a continuous increase in OTULIN expression within 72 h, and OTULIN expression was increased in activated microglial cells. OTULIN overexpression obviously decreased the cerebral infarct volume, improved the neurological function deficits, and reduced neuronal loss at 72 h after reperfusion, and it also inhibited the activation of microglia and attenuated the release of TNF-?, IL-1?, and IL-6 by suppressing the NF-?B pathway at 24 h after tMCAO. In vitro, OTULIN overexpression inhibited the microglia-mediated neuroinflammation by reducing the production of TNF-?, IL-1?, and IL-6 via depressing the NF-?B pathway in both PM and N9 cells. CONCLUSIONS:OTULIN provides a potential therapeutic target for ischemic brain injury by ameliorating the excessive activation of microglial cells and neuroinflammation through repressing the NF-?B signaling pathway.
Project description:Fatty acid-binding proteins (FABPs) regulate the intracellular dynamics of fatty acids, mediate lipid metabolism and participate in signaling processes. However, the therapeutic efficacy of targeting FABPs as novel therapeutic targets for cerebral ischemia is not well established. Previously, we synthesized a novel FABP inhibitor, i.e., FABP ligand 6 [4-(2-(5-(2-chlorophenyl)-1-(4-isopropylphenyl)-1H-pyrazol-3-yl)-4-fluorophenoxy)butanoic acid] (referred to here as MF6). In this study, we analyzed the ability of MF6 to ameliorate transient middle cerebral artery occlusion (tMCAO) and reperfusion-induced injury in mice. A single MF6 administration (3.0 mg/kg, per os) at 0.5 h post-reperfusion effectively reduced brain infarct volumes and neurological deficits. The protein-expression levels of FABP3, FABP5 and FABP7 in the brain gradually increased after tMCAO. Importantly, MF6 significantly suppressed infarct volumes and the elevation of FABP-expression levels at 12 h post-reperfusion. MF6 also inhibited the promotor activity of FABP5 in human neuroblastoma cells (SH-SY5Y). These data suggest that FABPs elevated infarct volumes after ischemic stroke and that inhibiting FABPs ameliorated the ischemic injury. Moreover, MF6 suppressed the inflammation-associated prostaglandin E<sub>2</sub> levels through microsomal prostaglandin E synthase-1 expression in the ischemic hemispheres. Taken together, the results imply that the FABP inhibitor MF6 can potentially serve as a neuroprotective therapeutic for ischemic stroke.
Project description:Emerging evidence indicates that methamphetamine (MA) abuse can impact cardiovascular disease. In humans, MA abuse is associated with an increased risk of stroke as well as an earlier age at which the stroke occurs. However, little is known about how chronic daily MA exposure can impact ischemic outcome in either humans or animal models. In the present study, mice were injected with MA (10 mg/kg, i.p.) or saline once daily for 10 consecutive days. Twenty-four hours after the final injection, mice were subjected to transient middle cerebral artery occlusion (tMCAO) for one hour followed by reperfusion. Mice were tested for novel object memory at 96 h post-reperfusion, just prior to removal of brains for quantification of infarct volume using 2,3,5-Triphenyltetrazolium Chloride (TTC) staining. Mice treated with MA prior to tMCAO showed decreased object memory recognition and increased infarct volume compared to saline-treated mice. These findings indicate that chronic MA exposure can worsen both cognitive and morphological outcomes following cerebral ischemia.