apelin-13-peptide and Infarction--Middle-Cerebral-Artery

The precise mechanisms whereby apelin-13 acts against ischemic stroke have remained in the dark. Hence, this study aims to examine the effects of apelin-13 on hypothalamic-pituitary-adrenal (HPA) axis over activation, Jak2-STAT3 signaling pathway, and inflammation following ischemic stroke.. Middle cerebral artery occlusion (MCAO) was used to induce the cerebral ischemic/reperfusion injury (I/RI). Thirty-five male Wistar rats (250-300 g, 8 weeks old) were randomly divided into sham, MCAO, and intravenous (IV) apelin-13 treated groups which received 10, 20, and 40 µg/kg 5 min before reperfusion (n = 7). Neurological status (modified Longa scoring scale), infarct volume, serum levels of malondialdehyde (MDA), total antioxidant capacity (TAC), interleukin 6 (IL-6), corticosterone, and the expressions of the Jak2/STAT3 were assessed.. Our results confirm that IV administration of all three doses of apelin-13 significantly improved neurological defects and reduced infarct volume following cerebral I/RI. Furthermore, we observed that acute stroke caused a rise in the expression of the Jak2/STAT3, IL-6, corticosterone, and MDA content, while apelin-13 could reduce the expression of the Jak2/STAT3 and the serum indices in a dose-dependent manner. The 40 µg/kg dose of apelin-13 was also more effective in reducing the infarct volume and improving TAC.. Our findings suggest that apelin-13 has protective effects against cerebral I/RI-related inflammation and also could attenuate the HPA axis over activation.

Topics: Animals; Brain Ischemia; Corticosterone; Hypothalamo-Hypophyseal System; Infarction, Middle Cerebral Artery; Inflammation; Interleukin-6; Ischemic Stroke; Janus Kinase 2; Male; Pituitary-Adrenal System; Rats; Rats, Wistar; Reperfusion Injury; Signal Transduction

It has been reported that apelin-13 possesses neuroprotective effects against cerebral ischemia/reperfusion injury (IRI). Disabilities in sense, movement and balance are the major stroke complications which, result in a high rate of mortality. Here, effects of intravenous (IV) injection of apelin-13 on the severity of neural death, infarct volume, neurological defects and its association with nitric oxide (NO) were investigated. A rat model of cerebral IRI was created by middle cerebral artery occlusion (MCAO) for 60 min and restoration of blood flow for 23 h. Animals were randomly assigned into six groups: sham, ischemia (MCAO), vehicle (MCAO + PBS) and three treatment groups (MCAO + apelin-13 in 10, 20, 40 μg/kg doses, IV). All injections were carried out via tail vein injection 5 min before reperfusion. Neural loss and infarct volume were evaluated by Nissl and 2,3,5-triphenyltetrazolium chloride (TTC) staining, respectively. Neurological defects were scored by standard modified criteria. Serum NO was measured by colorimetric method. Apelin-13 in doses of 20 and 40 μg/kg significantly reduced neural death, infarct volume and disturbance of sensory-motor balance compared to control and vehicle groups (p < 0.05). Serum NO levels reduced in MCAO groups compared to sham. Apelin-13 restored serum NO levels at 20 μg/kg dose (p < 0.05). Our data showed beneficial effect of IV injection of apelin-13 on sensory-motor balance defects by reducing neural death and restoration of serum NO levels. The present study shows the validity of apelin-13 in treatment of ischemic stroke in different administration methods.

Topics: Animals; Disease Models, Animal; Infarction, Middle Cerebral Artery; Injections, Intravenous; Intercellular Signaling Peptides and Proteins; Male; Motor Skills; Neuroprotective Agents; Nitric Oxide; Rats; Rats, Wistar; Reperfusion Injury

Oxidative stress, an adverse consequence of brain ischemia-reperfusion injury (IRI), activates matrix metalloproteinase enzymes which cause to destruction of extracellular matrix and tight junction proteins. Oxidative stress during stroke increases serum endothelin-1 and endothelin B receptor (ETBR) expression. Apelin-13, an endogenous peptide, is expressed in numerous tissues that regulate diverse physiological and pathological processes. This study aimed to investigate the effect of intravenous (IV) injection of apelin-13 on cerebral vasogenic edema due to brain IRI. Animals were divided into sham, ischemia, and treat groups. IRI model was induced by middle cerebral artery occlusion (MCAO) for 60 min followed by 23 h reperfusion. Apelin-13 was injected into the tail vein 5 min before reperfusion. Neurological defects were evaluated with longa test. Brain water content and BBB permeability were assessed according to cerebral dry-wet weight and brain Evans blue extraction. Malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were measured using the colorimetric method. Expression of occludin and claudin-5, matrix metalloproteinase- 2 and 9 (MMP-2 & 9) and, ETBR were evaluated using Western blot. Brain IRI was associated with BBB breakdowns and vasogenic edema. Apelin-13 significantly reduced BBB permeability and vasogenic edema. Apelin-13 significantly attenuated IRI-related oxidative stress. Apelin-13 decreased expression of mmp-2, 9 and ETBR, prevented from decrement of occludin and claudin-5 expersion, which protected BBB integrity and reduced vasogenic edema. In conclusion, our results have suggested that an IV injection of apelin-13 could somehow reduce vasogenic edema via targeting oxidative stress and ETBR expression.

Topics: Animals; Antioxidants; Brain Chemistry; Brain Edema; Claudin-5; Infarction, Middle Cerebral Artery; Infusions, Intravenous; Intercellular Signaling Peptides and Proteins; Ischemic Stroke; Male; Matrix Metalloproteinase Inhibitors; Occludin; Oxidative Stress; Rats; Rats, Wistar; Receptor, Endothelin B

Previous studies had showed that Apelin 13 could protect against apoptosis induced by ischemic/reperfusion (I/R). However, the mechanisms whereby Apelin 13 protected brain I/R remained to be elucidated. The present study was designed to determine whether Apelin 13 provided protection through AMPK/GSK-3β/Nrf2 pathway.. In vivo, the I/R model was induced and Apelin 13 was given intracerebroventricularly 15 min before reperfusion. The neurobehavioral scores, infarction volumes, and some cytokines in the brain were measured. For in vitro study, PC12 cells were used. To clarify the mechanisms, proteases inhibitors or siRNA were used. Protein levels were investigated by western blotting.. The results showed that Apelin 13 treatment significantly reduced infarct size, improved neurological outcomes, decreased brain edema, and inhibited cell apoptosis, oxidative stress, and neuroinflammation after I/R. Apelin 13 significantly increased the expression of Nrf2 and the phosphorylation levels of AMPK and GSK-3β. Furthermore, in cultured PC12 cells, the same protective effects were also observed. Silencing Nrf2 gene with its siRNA abolished the Apelin 13's prevention of I/R-induced PC12 cell injury, oxidative stress, and inflammation. Inhibition of AMPK by its siRNA decreased the level of Apelin 13-induced Nrf2 expression and diminished the protective effects of Apelin 13. The interplay relationship between GSK-3β and Nrf2 was also verified with relative overexpression. Using selective inhibitors, we further identified the upstream of AMPK/GSK-3β/Nrf2 is AR/Gα/PLC/IP3/CaMKK.. In conclusion, the previous results showed that Apelin 13 protected against I/R-induced ROS-mediated inflammation and oxidative stress through activating the AMPK/GSK-3β pathway by AR/Gα/PLC/IP3/CaMKK signaling, and further upregulated the expression of Nrf2-regulated antioxidant enzymes.

Topics: Animals; Brain Ischemia; Glycogen Synthase Kinase 3 beta; Infarction, Middle Cerebral Artery; Injections, Intraventricular; Intercellular Signaling Peptides and Proteins; Male; Mitogen-Activated Protein Kinases; Neuroprotective Agents; NF-E2-Related Factor 2; Oxidative Stress; PC12 Cells; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Signal Transduction; Stroke

Apelin-13 has been found to have protective effects on many neurological diseases, including cerebral ischemia. However, whether Apelin-13 acts on blood-brain barrier (BBB) disruption following cerebral ischemia is largely unknown. Aquaporin-4 (AQP4) has a close link with BBB due to the high concentration in astrocyte foot processes and regulation of astrocytes function. Here, we aimed to test Apelin-13's effects on ischemic BBB injury and examine whether the effects were dependent on AQP4.. We detected the expression of AQP4 induced by Apelin-13 injection at 1, 3, and 7 days after middle cerebral artery occlusion. Meanwhile, we examined the effects of Apelin-13 on neurological function, infarct volume, and BBB disruption owing to cerebral ischemia in wild type mice, and tested whether such effects were AQP4 dependent by using AQP4 knock-out mice. Furthermore, we assessed the possible signal transduction pathways activated by Apelin-13 to regulate AQP4 expression via astrocyte cultures.. It was found that Apelin-13 highly increased AQP4 expression as well as reduced neurological scores and infarct volume. Importantly, Apelin-13 played a role of BBB protection in both types of mice by reducing BBB permeability, increased vascular endothelial growth factor, upregulated endothelial nitric oxide synthase, and downregulated inducible NOS. In morphology, we demonstrated Apelin-13 suppressed tight junction opening and endothelial cell swelling via electron microscopy detection. Meanwhile, Apelin-13 also alleviated apoptosis of astrocytes and promoted angiogenesis. Interestingly, effects of AQP4 on neurological function and infarct volume varied with time course, while AQP4 elicited protective effects on BBB at all time points. Statistical analysis of 2-way analysis of variance with replication indicated that AQP4 was required for these effects. In addition, Apelin-13 upregulated phosphorylation of extracellular signal-regulated kinase (ERK) and Akt as well as AQP4 protein in cultured astrocytes. The latter was inhibited by ERK and phosphatidylinositol 3'-kinase (PI3K) inhibitors.. Our data suggest that Apelin-13 protects BBB from disruption after cerebral ischemia both morphologically and functionally, which is highly associated with the increased levels of AQP4, possibly through the activation of ERK and PI3K/Akt pathways. This study provides double targets to protection of ischemic BBB damage, which can present new insights to drugs development.

Topics: Animals; Apoptosis; Aquaporin 4; Astrocytes; Blood-Brain Barrier; Capillary Permeability; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Extracellular Signal-Regulated MAP Kinases; Infarction, Middle Cerebral Artery; Intercellular Signaling Peptides and Proteins; Male; Mice, Knockout; Neovascularization, Physiologic; Neuroprotective Agents; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Tight Junctions; Time Factors; Vascular Endothelial Growth Factor A

Apelin has been proved to be protective against apoptosis induced by ischemic reperfusion. However, mechanisms whereby apelin produces neuroprotection remain to be elucidated. AMP-activated protein kinase (AMPK) is a master energy sensor that monitors levels of key energy metabolites. It is activated via AMPKαThr172 phosphorylation during cerebral ischemia and appears to be neuroprotective. In this study, we investigated the effect of apelin on AMPKα and tested whether apelin protecting against apoptosis was associated with AMPK signals. Focal transient cerebral ischemia/reperfusion (I/R) model in male ICR mice was induced by 60 min of ischemia followed by reperfusion. Apelin-13 was injected intracerebroventricularly 15 min before reperfusion. AMPK inhibitor, compound C, was injected to mice intraperitoneally at the onset of ischemia. In experiment 1, the effect of apelin-13 on AMPKα was measured. In experiment 2, the relevance of AMPKα and apelin-13' effect on apoptosis was measured. Data showed that apelin-13 significantly increased AMPKα phosphorylation level after cerebral I/R. Apelin-13, with the co-administration of saline, reduced apoptosis cells, down-regulated Bax and cleaved-caspase3 and up-regulated Bcl2. However, with the co-administration of compound C, apelin-13 was inefficient in affecting apoptosis and Bax, Bcl2 and cleaved-caspase3. The study provided the evidence that apelin-13 up-regulated AMPKα phosphorylation level in cerebral ischemia insults and AMPK signals participated in the mechanism of apelin-mediated neuroprotection.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; bcl-2-Associated X Protein; Enzyme Activation; Infarction, Middle Cerebral Artery; Intercellular Signaling Peptides and Proteins; Male; Mice, Inbred ICR; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Apelin-13 has protective effects on many neurological diseases, including cerebral ischemia. Here, we aimed to test Apelin-13's effects on ischemic neurovascular unit (NVU) injuries and investigate whether the effects were dependent on vascular endothelial growth factor (VEGF). We detected the expression of VEGF and its receptors (VEGFRs) induced by Apelin-13 injection at 1d, 3d, 7d and 14d after middle cerebral artery occlusion (MCAO). Meanwhile, we examined the effects of Apelin-13 on NVU in both in vivo and in vitro experiments as well as whether the effects were VEGF dependent by using VEGF antibody. We also assessed the related signal transduction pathways via multiple inhibitors. We demonstrated Apelin-13 highly increased VEGF and VEGFR-2 expression, not VEGFR-1. Importantly, Apelin-13 led to neurological functions improvement by associating with promotion of angiogenesis as well as reduction of neuronal death and astrocyte activation, which was markedly blocked by VEGF antibody. In cell cultures, Apelin-13 protected neurons, astrocytes and endothelial cells against oxygen-glucose deprivation (OGD) injuries. Moreover, the effect of Apelin-13 to up-regulate VEGF was suppressed by extracellular signal-regulated kinase (ERK) inhibitor U0126 and phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002. Our data suggest protective effects of Apelin-13 on ischemic NVU injuries are highly associated with the increase of VEGF binding to VEGFR-2, possibly acting through activation of ERK and PI3K/Akt pathways.

Topics: Animals; Astrocytes; Brain Ischemia; Cell Survival; Infarction, Middle Cerebral Artery; Intercellular Signaling Peptides and Proteins; Male; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

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.

Topics: Animals; Anti-Inflammatory Agents; Apelin Receptors; Calcium-Binding Proteins; Cerebral Cortex; Drug Evaluation, Preclinical; Glial Fibrillary Acidic Protein; HMGB1 Protein; Infarction, Middle Cerebral Artery; Injections, Intraventricular; Intercellular Signaling Peptides and Proteins; Male; Microfilament Proteins; Neuroprotective Agents; Peroxidase; Rats, Wistar; Receptors, G-Protein-Coupled

The adipocytokine apelin is a peptide that was isolated from a bovine stomach for the first time. This peptide and its receptor are abundantly expressed in the nervous and cardiovascular systems. According to previous studies, apelin-13 protects cardiomyocytes from ischemic injury as well as apoptosis. In addition, this peptide has a neuroprotective effect on hippocampal and cultured mouse cortical neurons against NMDA receptor-mediated excitotoxicity. The present study was conducted to determine whether apelin-13 provides protection in transient focal cerebral ischemia. Focal ischemia was induced by 60-min middle cerebral artery occlusion (MCAO), followed by 23-h reperfusion. Saline as a vehicle and apelin-13 at doses of 25, 50, and 100 μg were injected intracerebroventriculary (ICV) at the beginning of ischemia. Infarct volume ,brain edema, motor dysfunction, and apoptosis were assessed 24 h after MCAO. Treatment with apelin-13 at doses of 50 and 100 μg ICV markedly reduced total infarct volumes by 45 and 55 %, respectively (P < 0.001), but injection of apelin at lower dose (25 μg) did not change infarct volume significantly (P > 0.05). In addition, apelin-13 at doses of 50 and 100 μg reduced brain edema (P < 0.001) and inhibited apoptosis by decreasing caspase-3 activation (P < 0.001). Apelin-13 did not significantly change neurological dysfunction (P > 0.05).

Topics: Animals; Apoptosis; Brain Edema; Brain Ischemia; Caspase 3; Cattle; Disease Models, Animal; Dose-Response Relationship, Drug; Infarction, Middle Cerebral Artery; Injections, Intraventricular; Intercellular Signaling Peptides and Proteins; Male; Neuroprotective Agents; Rats; Rats, Wistar; Severity of Illness Index