Yanyou Pan

Neuroprotective effects of apelin-13 on experimental ischemic stroke through suppression of inflammation

Acute inflammation plays an important role in the pathogenic progression of post-ischemic neuronal damage. Apelin-13 has been investigated as a neuropeptide for various neurological disorders. The present study was performed to evaluate the effects of apelin-13 on the inflammation of cerebral ischemia/reperfusion (I/R) injury. Transient focal I/R model in male Wistar rats were induced by 2h middle cerebral artery occlusion (MCAO) followed by 24h reperfusion. Rats then received treatment with apelin-13 or vehicle after ischemia at the onset of reperfusion. The neurological deficit was evaluated and the infarct volume was measured by TTC staining. The activity of myeloperoxidase (MPO) was measured. The expression of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and intercellular adhesion molecule-1 (ICAM-1) were measured using real-time PCR. And the expression of apelin receptor (APJ), ionized calcium-binding adapter molecule-1 (Iba1), glial fibrillary acidic protein (GFAP) and high mobility group box 1 (HMGB1) were measured by immunohistochemistry and western blot. Our results demonstrated that treatment with apelin-13 in I/R rats markedly reduced neurological deficits and the infarct volume. The increase of MPO activity induced by I/R was inhibited by apelin-13 treatment. The real-time PCR showed that apelin-13 decreased the expression of inflammatory cytokines such as IL-1β, TNF-α and ICAM-1 in I/R rats. The expression of APJ in I/R rats was increased. And the expression of Iba1, GFAP and HMGB1 in I/R rats was decreased by apelin-13 treatment indicating the inhibition of microglia, astrocytes and other inflammatory cells. In conclusion, apelin-13 is neuroprotective for neurons against I/R through inhibiting the neuroinflammation.

Orexin receptors exert a neuroprotective effect in Alzheimer’s disease (AD) via heterodimerization with GPR103

Orexins are neuropeptides that regulate the sleep-wake cycle and feeding behaviour. QRFP is a newly discovered neuropeptide which exerts similar orexigenic activity, thus playing an important role in energy homeostasis and regulation of appetite. The exact expression and signalling characteristics and physiological actions of QRFP and its receptor GPR103 are poorly understood. Alzheimer’s disease (AD) patients experience increased nocturnal activity, excessive daytime sleepiness, and weight loss. We hypothesised therefore that orexins and QRFP might be implicated in the pathophysiology of AD. We report that the down-regulation of hippocampal orexin receptors (OXRs) and GPR103 particularly in the cornu ammonis (CA) subfield from AD patients suffering from early onset familial AD (EOFAD) and late onset familial AD (LOAD). Using an in vitro model we demonstrate that this downregulation is due to to Aβ-plaque formation and tau hyper-phosphorylation. Transcriptomics revealed a neuroprotective role for both orexins and QRFP. Finally we provide conclusive evidence using BRET and FRET that OXRs and GPR103 form functional hetero-dimers to exert their effects involving activation of ERK1/2. Pharmacological intervention directed at the orexigenic system may prove to be an attractive avenue towards the discovery of novel therapeutics for diseases such as AD and improving neuroprotective signalling pathways.

Edaravone protected PC12 cells against MPP(+)-cytoxicity via inhibiting oxidative stress and up-regulating heme oxygenase-1 expression

Oxidative stress is involved in the pathogenesis of Parkinsons disease (PD). Edaravone has been shown to have a neuroprotective effect. In the present work, we investigated the effect of edaravone on 1-methyl-4-phenylpyridinium (MPP(+))-treated PC12 cells. Edaravone inhibited the decrease of cell viability and apoptosis induced by MPP(+) in PC12 cells. In addition, edaravone alleviated intracellular reactive oxygen species (ROS) production. MPP(+) induced heme oxygenase-1 (HO-1) expression, which was further enhanced by edaravone. The inhibitor of HO-1 zinc protoporphyrin-IX attenuated the neuroprotection of edaravone. So edaravone protected PC12 cells against MPP(+)-cytoxicity via inhibiting oxidative stress and up-regulating HO-1 expression. The data showed that edaravone was neuroprotective and could be potentially therapeutics for PD in future.

Identification of Conserved and Novel microRNAs in Cerebral Ischemia-Reperfusion Injury of Rat Using Deep Sequencing

MicroRNAs are a class of noncoding small RNAs that regulate gene expression by inhibiting target genes at post-transcriptional levels. MicroRNAs have been highlighted in many organs and tissues, including the brain. To identify special microRNAs involved in ischemia-reperfusion injury, we performed a comprehensive small RNA profiling in rat model and the control using Illumina high-throughput sequencing. A total of 9,444,562 and 10,290,391 clean reads were sequenced from two small RNA libraries constructed, respectively. Three hundred fifty-eight known microRNAs were identified, in which 78 microRNAs exhibited significantly differential expression between model and control. In addition, 62 and 68 novel miRNAs were found in model and control, respectively. Comparative analysis showed that 24 novel microRNAs were differentially expressed with greater than six-fold change. The GO annotation suggested that predicted targets of microRNAs were enriched into the category of metabolic process, cell part, cell-extracellular communications, and so on. KEGG pathway analysis suggested that these genes were involved in many important pathways, mainly including signaling transduction, MAPK signaling pathway, NF-κB signaling pathway, and neurotrophin signaling pathway. Our findings provided a deeper understanding to the regulatory mechanism of microRNAs underlying cerebral ischemia, therefore benefitting the improvement of the protection and treatment strategies of this disease.

Global gene expression profile of cerebral ischemia-reperfusion injury in rat MCAO model

It is well-established that reperfusion following cerebral ischemic injury gives rise to secondary injury accompanied by structural and functional damage. However, it remains unclear how global genes changes in cerebral ischemia-reperfusion injury (IRI). This study investigated global gene expression in the hippocampi of Wistar rats following transient cerebral IRI using an RNA-sequencing strategy. The results revealed ≥2-fold up-regulation of 156 genes and ≥2-fold down-regulation of 26 genes at 24 h post-reperfusion. Fifteen differentially expressed genes were selected to confirm the RNA-sequencing results. Gene expression levels were dynamic, with the peak expression level of each gene occurring at different time points post-reperfusion. Gene Ontology (GO) analysis classified the differentially expressed genes as mainly involved in inflammation, stress and immune response, glucose metabolism, proapoptosis, antiapoptosis, and biological processes. KEGG pathway analysis suggested that IRI activated different signaling pathways, including focal adhesion, regulation of actin cytoskeleton, cytokine-cytokine receptor interaction, MAPK signaling, and Jak-STAT signaling. This study describes global gene expression profiles in the hippocampi of Wistar rats using the middle cerebral artery occlusion (MCAO) model. These findings provide new insights into the molecular pathogenesis of IRI and potential drug targets for the prevention and treatment of IRI in the future.

The Orexin/Receptor System: Molecular Mechanism and Therapeutic Potential for Neurological Diseases

Orexins, also known as hypocretins, are two neuropeptides secreted from orexin-containing neurons, mainly in the lateral hypothalamus (LH). Orexins orchestrate their effects by binding and activating two G-protein–coupled receptors (GPCRs), orexin receptor type 1 (OX1R) and type 2 (OX2R). Orexin/receptor pathways play vital regulatory roles in many physiological processes, especially feeding behavior, sleep–wake rhythm, reward and addiction and energy balance. Furthermore several reports showed that orexin/receptor pathways are involved in pathological processes of neurological diseases such as narcolepsy, depression, ischemic stroke, drug addiction and Alzheimer’s disease (AD). This review article summarizes the expression patterns, physiological functions and potential molecular mechanisms of the orexin/receptor system in neurological diseases, providing an overall framework for considering these pathways from the standpoints of basic research and clinical treatment of neurological diseases.

Neuroprotection of bradykinin/bradykinin B2 receptor system in cerebral ischemia

Bradykinin B2 receptor (B2R) activated by its endogenous ligand bradykinin participates in various physiological processes including neurogenesis, neuronal differentiation, and control of inflammation and blood pressure. Besides these effects, B2R has been demonstrated to protect neurons from ischemia/reperfusion (I/R) injury. Here, we highlight the mechanisms of BK/B2R-mediated neuroprotective effects in the peripheral and central nervous systems. Moreover, this review article summarizes some of the signaling pathways of B2R in cerebral ischemia, leading to a better understanding of its neuroprotection.

Ghrelin Through GHSR1a and OX1R Heterodimers Reveals a Gαs–cAMP-cAMP Response Element Binding Protein Signaling Pathway in Vitro

Growth hormone secretagogue receptor 1α (GHSR1a) and Orexin 1 receptor (OX1R) are involved in various important physiological processes, and have many similar characteristics in function and distribution in peripheral tissues and the central nervous system. We explored the possibility of heterodimerization between GHSR1a and OX1R and revealed a signal transduction pathway mechanism. In this study, bioluminescence and fluorescence resonance energy transfer and co-immunoprecipitation (Co-IP) analyses were performed to demonstrate the formation of functional GHSR1a/OX1R heterodimers. This showed that a peptide corresponding to the 5-transmembrane domain of OX1R impaired heterodimer construction. We found that ghrelin stimulated GHSR1a/OX1R heterodimer cells to increase the activation of Gαs protein, compared to the cells that express GHSR1a. Stimulation of GHSR1a/OX1R heterodimers with orexin-A did not alter GPCR interactions with Gα protein subunits. GHSR1a/OX1R heterodimers induced Gαs and downstream signaling pathway activity, including increase of cAMP-response element luciferase reporter activity and cAMP levels. In addition, ghrelin induced a higher proliferation rate in SH-SY5Y cells than in controls. This suggests that ghrelin GHSR1a/OX1R heterodimers promotes an upregulation of a Gαs-cAMP-cAMP-responsive element signaling pathway in vitro and an increase in neuroblastoma cell proliferation.

RNA-seq expression profiling of rat MCAO model following reperfusion Orexin-A

Orexin-A is a neuropeptide with potent neuroprotective activity towards cerebral ischemia-reperfusion (I/R) injury, but few studies have attempted to elucidate the mechanism. Herein, we performed global gene expression profiling of the hippocampus following reperfusion with Orexin-A using RNA sequencing (RNA-seq). RNA-seq identified 649 differentially expressed genes (DEGs) in the Orexin-A group compared with saline controls (I/R group), of which 149 were up-regulated and 500 were down-regulated. DEGs were confirmed using qRT-PCR, their molecular functions, biological processes and molecular components were explored using Gene Ontology (GO) analysis and 206 KEGG pathways were associated with Orexin-A treatment. MAPK, chemokine and calcium signalling pathways were mainly responsible for the neuroprotective effects of Orexin-A. Hspb1, Igf2 and Ptk2b were selected for functional interaction analysis by GeneMANIA. The results suggest that Orexin-A modifies gene expression in the hippocampus, leading to neuroprotection from I/R injury. The study provides a basis for future elucidation of the molecular mechanisms underlying Orexin-A.

C-terminus of OX2R significantly affects downstream signaling pathways

The human orexin 2 receptor (OX2R) is a G-protein-coupled receptor (GPCR) that has been implicated in a number of diverse physiological functions. Recent studies have identified a number of functions of the C-termini of GPCRs. However, the importance of the OX2R C-terminus in regulating signaling and surface expression remains unclear. In the present study, the function of the OX2R C-terminus was investigated using three C-terminal mutants, which were truncated at residues 368, 384 and 414, respectively, and the wild-type control, which expressed the full-length OX2R. HEK-293 cells were transfected with the mutated and control OX2R constructs. ELISA, western blot analysis and calcium assays were used to investigate the effects of the mutations on OX2R function. The present results demonstrated that residues 385–414 and 415–444 exhibited a cumulative effect on the surface expression of OX2R. Residues 369–384 exhibited a significant influence on inositol phosphate production and extracellular signal-regulated kinase 1/2 phosphorylation. Residues 385–414 significantly influenced agonist-induced internalization, whereas residues 369–384 and 385–414 significantly influenced Ca2+ release. The results of the present study suggest that the C-terminus of OX2R is important for its role in various physiological and pathological processes, and may therefore be associated with such disorders as depression and anorexia.