Apelin-36, a potent peptide,protects against ischemic brain injury by activating the PI3K/Akt pathway

Apelin is an endogenous ligand of G protein-coupled receptor-apelin and angiotensin-1-like receptor (APJ). The biological effects of apelin–APJ system are reported in multiple systems including cardiovascular, endocrinal, and gastrointestinal system. Previous studies had shown that apelin-13 is a potential protective agent on cardiac ischemia; however, the role of apelin in the central nervous system remained unknown. In this study, we investigated therapeutic effects of apelin-36, a long form of apelin, in ischemic brain injury models. We found that apelin-36 reduced cerebral infarct volume in the middle cerebral artery occlusion (MCAO) model and the neonatal hypoxic/ischemic (H/I) injury model. Apelin-36 improved neurological deficits in the MCAO model and promoted long-term functional recovery after H/I brain injury. We further explored the protective mechanisms of apelin-36 on H/I brain injury. We clearly demonstrated that apelin-36 significantly reduced the levels of cleaved caspase-3 and Bax, two well-established apoptotic markers after H/I injury, indicating the anti-apoptotic activity of apelin-36 in ischemic injury. Since apelin-36 increased the level of phosphorylated Akt after H/I injury, we treated neonates with a specific PI3K inhibitor LY294002. We found that LY294002 decreased the phosphorylated Akt level and attenuated protective effects of apelin-36 on apoptosis. These suggested that the PI3K/Akt pathway was at least in part involved in the anti-apoptotic mechanisms of apelin-36. Our findings demonstrated that apelin-36 was a promising therapeutic agent on the treatment of ischemic brain injury.     

Protective effect of apelin on cultured rat bone marrow mesenchymal stem cells against apoptosis

Bone marrow-derived mesenchymal stem cells (BMSCs) have shown great promise for ischemic tissue repair. However, poor viability of transplanted BMSCs within ischemic tissues has limited their therapeutic potential. Apelin, an endogenous peptide, whose level is elevated following ischemia, has been shown to enhance survival of cardiomyocytes and neuronal cells during ischemia. We hypothesized that apelin-13 protects BMSCs from apoptotic death. In this paper we determined the potential mechanism of apelin-13 effects using cultured BMSCs from adult rats. Apoptosis was induced by the specific apoptotic insult serum deprivation (SD) for up to 36 h. Apoptotic cell death was measured using immunostaining and Western blotting in the presence and absence of apelin-13 (0.1 to 5.0 nM) co-applied during SD exposure. SD-induced apoptosis was significantly reduced by apelin-13 in a concentration-dependent manner. SD-induced mitochondrial depolarization, cytochrome c release, and caspase-3 activation were largely prevented by apelin-13. The apelin-13 anti-apoptotic effects were blocked by inhibiting the MAPK/ERK1/2 and PI3K/Akt signaling pathways. Taken together, our findings indicate that apelin-13 is a survival factor for BMSCs and its anti-apoptotic property may prove to be of therapeutic significance in terms of exploiting BMSC-based transplantation therapy.     

Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons

The adipocytokine apelin and its G protein-coupled APJ receptor were initially isolated from a bovine stomach and have been detected in the brain and cardiovascular system. Recent studies suggest that apelin can protect cardiomyocytes from ischemic injury. Here, we investigated the effect of apelin on apoptosis in mouse primary cultures of cortical neurons. Exposure of the cortical cultures to a serum-free medium for 24 h induced nuclear fragmentation and apoptotic death; apelin-13 (1.0-5.0 nM) markedly prevented the neuronal apoptosis. Apelin neuroprotective effects were mediated by multiple mechanisms. Apelin-13 reduced serum deprivation (SD)-induced ROS generation, mitochondria depolarization, cytochrome c release and activation of caspase-3. Apelin-13 prevented SD-induced changes in phosphorylation status of Akt and ERK1/2. In addition, apelin-13 attenuated NMDA-induced intracellular Ca²⁺ accumulation. These results indicate that apelin is an endogenous neuroprotective adipocytokine that may block apoptosis and excitotoxic death via cellular and molecular mechanisms. It is suggested that apelins may be further explored as a potential neuroprotective reagent for ischemia-induced brain damage.     

Neuroprotective Effect of Fucoidan on H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Apoptosis in PC12 Cells Via Activation of PI3K/Akt Pathway

One of the plausible ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity. In this study, we investigated the neuroprotective effect of fucoidan on H(2)O(2)-induced apoptosis in PC12 cells and the possible signaling pathways involved. The results showed that fucoidan inhibited the decrease of cell viability, scavenged ROS formation and reduced lactate dehydrogenase release in H(2)O(2)-induced PC12 cells. These changes were associated with an increase in superoxide dismutase and glutathione peroxidase activity, and reduction in malondialdehyde. In addition, fucoidan treatment inhibited apoptosis in H(2)O(2)-induced PC12 cells by increasing the Bcl-2/Bax ratio and decreasing active caspase-3 expression, as well as enhancing Akt phosphorylation (p-Akt). However, the protection of fucoidan on cell survival, p-Akt, the Bcl-2/Bax ratio and caspase-3 activity were abolished by pretreating with phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. In consequence, fucoidan might protect the neurocytes against H(2)O(2)-induced apoptosis via reducing ROS levels and activating PI3K/Akt signaling pathway.     

RUNX1对氧糖剥夺诱导PC12细胞凋亡的保护作用研究

目的:研究RUNX1在PC12细胞氧糖剥夺模型中的表达及其对PC12细胞的保护作用,并探讨其相关机制。方法:体外培养PC12细胞并构建氧糖剥夺模型,将细胞分为对照组、氧糖剥夺组、RUNX1 si RNA处理组、si RNA对照处理组(sicontrol)、pc DNA3.1-RUNX1处理组(pc RUNX1)和pc DNA3.1对照处理组(pc DNA 3.1)。q RT-PCR和western blot检测RUNX1、磷酸化Akt(p-Akt)和总Akt(t-Akt)表达水平;MTT法检测细胞存活率;Annexin V-FITC/PI双染法检测细胞凋亡。结果:与对照组比较,RUNX1在PC12细胞氧糖剥夺模型中表达水平显著升高;沉默RUNX1可下调PC12细胞的存活率,促进细胞的凋亡,有效抑制p-Akt蛋白表达,而过表达RUNX1显著提高细胞存活率,抑制细胞凋亡,并上调p-Akt蛋白表达;此外,PI3K/Akt通路抑制剂LY294002明显抑制RUNX1过表达对细胞存活率的促进作用和对细胞凋亡的抑制作用。结论:RUNX1可通过PI3K/Akt信号通路保护OGD对PC12细胞的损伤作用。     

Quercetin regulates insulin like growth factor signaling and induces intrinsic and extrinsic pathway mediated apoptosis in androgen independent prostate cancer cells (PC-3)

Progression of prostate cancer is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. To investigate the effect of quercetin on insulin-like growth factor signaling and apoptosis in androgen independent prostate cancer cells (PC-3), IGF-IR, PI-3K, p-Akt, Akt, cyclin D1, Bad, cytochrome c, PARP, caspases-9 and 10 protein levels were assessed by western blot analysis. Mitochondrial membrane potency was detected by rhodamine-123 staining. Quercetin induced caspase-3 activity assay was performed for activation of apoptosis. Further, RT-PCR was also performed for Bad, IGF-I, II, IR, and IGFBP-3 mRNA expression. Quercetin significantly increases the proapoptotic mRNA levels of Bad, IGFBP-3 and protein levels of Bad, cytochrome C, cleaved caspase-9, caspase-10, cleaved PARP and caspase-3 activity in PC-3 cells. IGF-IRβ, PI3K, p-Akt, and cyclin D1 protein expression and mRNA levels of IGF-I, II and IGF-IR were decreased significantly. Further, treatment with PI3K inhibitor (LY294002) and quercetin showed decreased p-Akt levels. Apoptosis is confirmed by loss of mitochondrial membrane potential in quercetin treated PC-3 cells. This study suggests that quercetin decreases the survival of androgen independent prostate cancer cells by modulating the expression of insulin-like growth factors (IGF) system components, signaling molecules and induces apoptosis, which could be very useful for the androgen independent prostate cancer treatment.     

Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways

The aim of this study was to investigate the effects of apelin on proliferation and apoptosis of mouse osteoblastic MC3T3-E1 cells. APJ was expressed in MC3T3-E1 cells. Apelin did not affect Runx2 expression, alkaline phosphatase (ALP) activity, osteocalcin and type I collagen secretion, suggesting that it has no effect on osteoblastic differentiation of MC3T3-E1 cells. However, apelin stimulated MC3T3-E1 cell proliferation and inhibited cell apoptosis induced by serum deprivation. Our study also shows that apelin decreased cytochrome c release and caspase-3, capase-8 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Apelin activated c-Jun N-terminal kinase (JNK) and Akt (phosphatidylinositol 3-kinase downstream effector), and the JNK inhibitor SP600125, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO) inhibited its effects on proliferation and serum deprivation-induced apoptosis. Furthermore, apelin protected against apoptosis induced by the glucocorticoid dexamethasone or TNF-alpha. Apelin stimulates proliferation and suppresses serum deprivation-induced apoptosis of MC3T3-E1 cells and these actions are mediated via JNK and PI3-K/Akt signaling pathways.     

Protection afforded by quercetin against H2O2-induced apoptosis on PC12 cells via activating PI3K/Akt signal pathway

Cell damage and apoptosis induced by oxidative stress have been involved in various neurodegenerative diseases. This study aims to explore the neuro-protective effects of quercetin on PC12 cells apoptosis induced by hydrogen peroxide (H₂O₂) and the underlying mechanisms. The cell viability was detected, as well as the production of reactive oxygen species (ROS), lactate dehydrogenase (LDH) leakage, and the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) of the cells in control, H₂O₂ and quercetin groups. It finally turned out that quercetin might protect PC12 cells against the negative effect of H₂O₂ by decreasing of LDH release, ROS concentration and MDA level and regaining the GSH-Px and SOD activities. To investigate the mechanism, LY294002 was introduced, the phosphatidylinositol-3-kinase (PI3K) inhibitor. Bax/Bcl-2 ratio and Akt phosphorylation (p-Akt) were examined by Western blot analysis. The data showed that LY294002 almost had the same effects with H₂O₂, which was also significantly reversed by quercetin could enhance Bax/Bcl-2 ratio and adjust the p-Akt expression, which indicated quercetin might protect PC12 cells against the negative effect of H₂O₂ via activating the PI3K/Akt signal pathway.     

Quercetin Attenuates Cell Apoptosis in Focal Cerebral Ischemia Rat Brain Via Activation of BDNF–TrkB–PI3K/Akt Signaling Pathway

Many studies have demonstrated that apoptosis play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. Neuroprotective effect of quercetin has been shown in a variety of brain injury models including ischemia/reperfusion. It is not clear whether BDNF?CTrkB?CPI3K/Akt signaling pathway mediates the neuroprotection of quercetin, though there has been some reports on the quercetin increased brain-derived neurotrophic factor (BDNF) level in brain injury models. We therefore first examined the neurological function, infarct volume and cell apoptosis in quercetin treated middle cerebral artery occlusion (MCAO) rats. Then the protein expression of BDNF, cleaved caspase-3 and p-Akt were evaluated in either the absence or presence of PI3K inhibitor (LY294002) or tropomyosin receptor kinase B (TrkB) receptor antagonist (K252a) by immunohistochemistry staining and western blotting. Quercetin significantly improved neurological function, while it decreased the infarct volume and the number of TdT mediated dUTP nick end labeling positive cells in MCAO rats. The protein expression of BDNF, TrkB and p-Akt also increased in the quercetin treated rats. However, treatment with LY294002 or K252a reversed the quercetin-induced increase of BDNF and p-Akt proteins and decrease of cleaved caspase-3 protein in focal cerebral ischemia rats. These results demonstrate that quercetin can decrease cell apoptosis in the focal cerebral ischemia rat brain and the mechanism may be related to the activation of BDNF?CTrkB?CPI3K/Akt signaling pathway.     

The apelin receptor is coupled to Gi1 or Gi2 protein and is differentially desensitized by apelin fragments

The apelin receptor is a G protein-coupled receptor to which two ligand fragments, apelin-(65-77) and apelin-(42-77), can bind. To address the physiological significance of the existence of dual ligands for a single receptor, we first compared the ability of the apelin fragments to regulate intracellular effectors, to promote G protein coupling, and to desensitize the response in Chinese hamster ovary cells expressing the murine apelin receptor. We found that both apelin fragments inhibited adenylyl cyclase and increased the phosphorylation of ERK or Akt. Using stably transfected cells expressing a pertussis toxin-insensitive alpha(i) subunit, we demonstrated that each apelin fragment promoted coupling of the apelin receptor to either Galpha(i1) or Galpha(i2) but not to Galpha(i3). Although preincubation with each apelin fragment induced a desensitization at the level of the three effectors, preincubation with apelin-(42-77) also increased basal effector activity. In addition, a C-terminal deletion of the apelin receptor decreased the desensitization induced by apelin-(65-77) but did not alter the desensitization pattern induced by apelin-(42-77). Finally, in umbilical endothelial cells, which we have recently shown to express the apelin receptor, the Galpha(i1) and Galpha(i2) subunits are also expressed, ERK and Akt phosphorylation is desensitized after preincubation with apelin-(65-77), and basal levels of Akt phosphorylation are increased after preincubation with apelin-(42-77). In summary, apelin fragments regulate the same effectors, via the preferential coupling of the apelin receptor to G(i1) or G(i2), but they promote a differential desensitization pattern that may be central to their respective physiological roles.     

臭椿酮诱导人黑色素瘤A375细胞凋亡及其机制研究

为研究臭椿酮(Ailanthone,AIL)诱导人黑色素瘤A375细胞凋亡的作用及作用机制,以人黑色素瘤A375细胞为研究对象,采用MTT法测定AIL对人黑色素瘤A375细胞生长增殖的抑制作用。用倒置相差显微镜观察AIL对A375细胞形态的影响,用荧光倒置显微镜观察Hoechst33258染色后AIL对A375细胞核的影响,用AnnexinV-FITC/PI双染法检测AIL诱导A375细胞凋亡的作用,用分光光度法检测caspase-3和caspase-9的活性,Westernblot检测p-PI3Kβ(Ser1070),PI3Kβ,p-Akt(Ser473)和Akt蛋白表达水平的变化,接着用PI3K抑制剂LY294002进行干预,进一步验证AIL对PI3K/Akt信号通路及细胞凋亡的影响。实验结果表明,AIL能够明显抑制A375细胞增殖,使A375细胞数目变少、附着力和透光性减弱,AIL能够诱导A375细胞凋亡,使其细胞核染色质发生固缩并呈现高亮,且使A375细胞早期及晚期凋亡率均增加,AIL作用后能够使caspase-3和caspase-9活性增加,AIL能够抑制PI3K和Akt蛋白磷酸化,从而使PI3K/Akt信号通路失活。较AIL单独作用,AIL和LY294002共同作用后对PI3K和Akt蛋白磷酸化的抑制作用增强且诱导凋亡作用增加,进一步说明AIL通过失活PI3K/Akt信号通路来诱导A375细胞凋亡。     

Protective Role Of Naringenin Against Aβ<Subscript>25-35</Subscript>-Caused Damage via ER and PI3K/Akt-Mediated Pathways

Senile plaque accumulation and neurofibrillary tangles are primary characteristics of Alzheimer’s disease. We aimed to assess the protective functions of naringenin against β-amyloid protein fragment 25-35 (Aβ_25-35)-caused nerve damage in differentiated PC12 cells, and study the potential mechanisms. We evaluated cell viability and apoptosis using the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test and flow cytometry, respectively. Moreover, we measured protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β), and caspase-3 activity via western blotting and RT-PCR. We found that naringenin protected cell against Aβ_25-35-caused nerve damage by increasing cell viability, promoting Akt and GSK3β activation, and inhibiting cell apoptosis and caspase-3 activity. However, treatment with the estrogen receptor (ER) antagonist ICI182, 780 or phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 suppressed the effects of naringenin. Our results suggested that naringenin could effectively suppress Aβ_25-35-caused nerve damage in PC12 cells by regulating the ER and PI3K/Akt pathways.     

Pharmacological Inhibition of PERK Attenuates Early Brain Injury After Subarachnoid Hemorrhage in Rats Through the Activation of Akt

Neuronal apoptosis is a central pathological process in subarachnoid hemorrhage (SAH)-induced early brain injury. Endoplasmic reticulum (ER) stress was reported to have a vital role in the pathophysiology of neuronal apoptosis in the brain. The present study was designed to investigate the potential effects of ER stress and its downstream signals in early brain injury after SAH. One hundred thirty-four rats were subjected to an endovascular perforation model of SAH. The RNA-activated protein kinase-like ER kinase (PERK) inhibitor GSK2606414 and the Akt inhibitor MK2206 were injected intracerebroventricularly. SAH grade, neurologic scores, and brain water content were measured 72 h after subarachnoid hemorrhage. Expression of PERK and its downstream signals, Akt, Bcl-2, Bax, and cleaved caspase-3, were examined using Western blot analysis. Specific cell types that expressed PERK were detected with double immunofluorescence staining. Neuronal cell death was demonstrated with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Our results showed that the expression of p-PERK and its downstream targets, p-eIF2α and ATF4, increased after SAH and peaked at 72 h after SAH. PERK was expressed mostly in neurons. The inhibition of PERK with GSK2606414 reduced p-PERK, p-eIF2α, and ATF4 expression. Furthermore, GSK2606414 treatment increased p-Akt levels and the Bcl-2/Bax ratio as well as decreased cleaved caspase-3 expression and neuronal death, thereby improving neurological deficits at 72 h after SAH. The selective Akt inhibitor MK2206 abolished the beneficial effects of GSK2606414. PERK, the major transducer of ER stress, is involved in neuronal apoptosis after SAH. The inhibition of PERK reduces early brain injury via Akt-related anti-apoptosis pathways. PERK may serve as a promising target for future therapeutic intervention.     

Miltirone Attenuates Reactive Oxygen Species-Dependent Neuronal Apoptosis in MPP+-Induced Cell Model of Parkinson’s Disease Through Regulating the PI3K/Akt Pathway

Miltirone is a phenanthrene-quinone derived from Salvia miltiorrhiza Bunge with anti-inflammatory and anti-oxidant effects. Our study aimed to explore the protective effect of miltirone on 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell model of Parkinson’s disease (PD). PharmMapper database was employed to predict the targets of miltirone. PD-related genes were identified using GeneCards database. The overlapping genes between miltirone and PD were screened out using Venn diagram. KEGG analysis was performed using DAVID and KOBAS databases. Cell viability, reactive oxygen species (ROS) generation, apoptosis, and caspase-3 activity were detected by CCK-8 assay, a ROS assay kit, TUNEL, and caspase-3 activity assay, respectively. Effect of miltirone on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway was explored by western blot analysis. A total of 214 targets of miltirone and 372 targets related to PD were attained, including 29 overlapping targets. KEGG analysis demonstrated that the 29 overlapping targets were both significantly enriched in the PI3K/Akt pathway. MPP⁺ stimulation reduced the cell viability in SH-SY5Y cells and neuronal primary cultures derived from human brain. Miltirone or N-acetylcysteine (NAC) attenuated MPP⁺-induced reduction in cell viability, ROS production, SOD activity reduction, apoptosis, and increase of caspase-3 activity. Additionally, miltirone recuperated MPP⁺-induced inactivation of the PI3K/Akt pathway. Moreover, treatment with LY294002, an inhibitor of the PI3K/Akt pathway, reversed the inhibitory effect of miltirone on MPP⁺-induced ROS generation and apoptosis in SH-SY5Y cells and neuronal primary cultures. In conclusion, miltirone attenuated ROS-dependent apoptosis in MPP⁺-induced cellular model of PD through activating the PI3K/Akt pathway.

     

Apelin-13 increases myocardial progenitor cells and improves repair postmyocardial infarction

Apelin is an endogenous ligand for the angiotensin-like 1 receptor (APJ) and has beneficial effects against myocardial ischemia-reperfusion injury. Little is known about the role of apelin in the homing of vascular progenitor cells (PCs) and cardiac functional recovery postmyocardial infarction (post-MI). The present study investigated whether apelin affects PC homing to the infarcted myocardium, thereby mediating repair and functional recovery post-MI. Mice were infarcted by coronary artery ligation, and apelin-13 (1 mg·kg(-1)·day(-1)) was injected for 3 days before MI and for 14 days post-MI. Homing of vascular PCs [CD133(+)/c-Kit(+)/Sca1(+), CD133(+)/stromal cell-derived factor (SDF)-1α(+), and CD133(+)/CXC chemokine receptor (CXCR)-4(+)] into the ischemic area was examined. Myocardial Akt, endothelial nitric oxide synthase (eNOS), VEGF, jagged1, notch3, SDF-1α, and CXCR-4 expression were assessed at 24 h and 14 days post-MI. Functional analyses were performed on day 14 post-MI. Mice that received apelin-13 treatment demonstrated upregulation of SDF-1α/CXCR-4 expression and dramatically increased the number of CD133(+)/c-Kit(+)/Sca1(+), CD133(+)/SDF-1α(+), and c-Kit(+)/CXCR-4(+) cells in infarcted hearts. Apelin-13 also significantly increased Akt and eNOS phosphorylation and upregulated VEGF, jagged1, and notch3 expression in ischemic hearts. This was accompanied by a significant reduction of myocardial apoptosis. Furthermore, treatment with apelin-13 promoted myocardial angiogenesis and attenuated cardiac fibrosis and hypertrophy together with a significant improvement of cardiac function at 14 days post-MI. Apelin-13 increases angiogenesis and improves cardiac repair post-MI by a mechanism involving the upregulation of SDF-1α/CXCR-4 and homing of vascular PCs.     

Apelin-13 protects the heart against ischemia-reperfusion injury through inhibition of ER-dependent apoptotic pathways in a time-dependent fashion

Endoplasmic reticulum (ER) stress is activated during and contributes to ischemia-reperfusion (I/R) injury. Attenuation of ER stress-induced apoptosis protects the heart against I/R injury. Using apelin, a ligand used to activate the apelin APJ receptor, which is known to be cardioprotective, this study was designed to investigate 1) the time course of changes in I/R injury after ER stress; 2) whether apelin infusion protects the heart against I/R injury via modulation of ER stress-dependent apoptosis signaling pathways; and 3) how phosphatidylinositol 3-kinase (PI3K)/Akt, endothelial nitric oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and ERK activation are involved in the protection offered by apelin treatment. The results showed that, using an in vivo rat I/R model induced by 30 min of ischemia followed by reperfusion, infarct size (IS) increased from 2 h of reperfusion (34.85 ± 2.14%) to 12 h of reperfusion (48.98 ± 3.35, P < 0.05), which was associated with an abrupt increase in ER stress-dependent apoptosis activation, as evidenced by increased CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, and JNK activation (CHOP: 2.49-fold increase, caspase-12: 2.09-fold increase, and JNK: 3.38-fold increase, P < 0.05, respectively). Administration of apelin at 1 μg/kg not only completely abolished the activation of ER stress-induced apoptosis signaling pathways at 2 h of reperfusion but also significantly attenuated time-related changes at 24 h of reperfusion. Using pharmacological inhibition, we also demonstrated that PI3K/Akt, AMPK, and ERK activation were involved in the protection against I/R injury via inhibition of ER stress-dependent apoptosis activation. In contrast, although eNOS activation played a role in decreasing IS at 2 h of reperfusion, it failed to modify either IS or ER stress-induced apoptosis signaling pathways at 24 h after reperfusion.     

Microvesicles-mediated communication between endothelial cells modulates,endothelial survival,and angiogenic function via transferring of miR-125a-5p

Endothelial cells (ECs) released microvesicles (EMVs) could modulate the functions of target cells by transferring their microRNAs (miRs). We have reported that miR-125a-5p protected EC function. In this study, we determined whether EMVs provided beneficial effects on ECs by transferring miR-125a-5p. Human brain microvessel ECs were transfected with miR-125a-5p mimic or miR-125a-5p short hairpin RNA to obtain miR-125a-5p overexpressing ECs and miR-125a-5p knockdown ECs, and their derived EMVs. For the functional study, ECs or hypoxia/reoxygenation injured ECs were coincubated with various EMVs. The survival and angiogenic function of ECs were measured. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used for measuring the levels of phosphoinositide 3-kinase (PI3K), phosphorylation-Akt (p-Akt)/Akt, p-endothelial nitric oxide synthase (p-eNOS), cleaved caspase-3, and miR-125a-5p. PI3K inhibitor was used for pathway analysis. EMVs promoted the proliferation, migration, and tube formation ability of ECs, and alleviated the apoptotic rate of ECs. These effects were associated by an increase in p-Akt/Akt and p-eNOS, and a decrease in cleaved caspase-3 could be abolished by LY294002. Overexpression or downregulation of miR-125a-5p in EMVs promoted or inhibited those effects of EMVs. EMVs could enhance the survival and angiogenic function of ECs via delivering miR-125a-5p to modulate the expression of PI3K/Akt/eNOS pathway and caspase-3.     

Apelin-13 attenuates cisplatin-induced cardiotoxicity through inhibition of ROS-mediated DNA damage and regulation of MAPKs and AKT pathways

Platinum-based chemotherapy represents one of the most effective ways in combating human cancers. However, the cardiotoxicity subsequent severely limited its clinical application. Increased evidences indicate that oxidative stress plays a crucial role in the pathological process of platinum-induced cardiotoxicity. It is reported that apelin-13 a bioactive peptide has the scavenging capacity of free radical, and it has the potential to regulate the cardiovascular system. Hence, the potential of apelin-13 to antagonize cisplatin-induced cardiotoxicity was evaluated in H9c2 rat myocardial cells in vitro and in C57 mice in vivo. The results showed that cisplatin indeed caused DNA damage in H9c2 cells by promoting the accumulation of intracellular reactive oxygen species (ROS) and superoxide anion, which led to cell apoptosis and resulted in overt cardiotoxicity. However, apelin-13 pre-treatment effectively attenuated the cisplatin-induced ROS and superoxide anion generation, inhibited DNA damage, and suppressed the PARP cleavage and caspases activation. Further investigation revealed that apelin-13 blocked cisplatin-induced H9c2 cells apoptosis involving the regulation of MAPKs and PI3K/Akt signaling pathway. Importantly, apelin-13 co-treatment also significantly attenuated cisplatin-induced cardiotoxicity in vivo by inhibiting myocardial cells apoptosis and improving angiogenesis in mice heart. Taken together, our results suggest that the use of apelin-13 may be an effective strategy for antagonizing the cardiotoxicity-induced by platinum-based chemotherapy.     

Apelin-13 protects against apoptosis by activating AMP-activated protein kinase pathway in ischemia stroke

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.     

Tetramethylpyrazine Protects Against Early Brain Injury and Inhibits the PERK/Akt Pathway in a Rat Model of Subarachnoid Hemorrhage

Neuronal apoptosis is a potentially fatal pathological process that occurs in early brain injury (EBI) after subarachnoid hemorrhage (SAH). There is an urgent need to identify effective therapeutics to alleviate neuronal apoptosis. Tetramethylpyrazine (TMP), as an important component of the Chinese traditional medicinal herb Ligusticum wallichii, has been widely used in China to treat cerebral ischemic injury and confer neuroprotection. In the present work, we investigate whether TMP can reduce EBI following SAH in rats, specifically via inactivating the PERK/Akt signaling cascade. One hundred twenty-five male Sprague–Dawley rats were used in the present study. TMP was administered by intravenous (i.v.) injection, and the Akt inhibitor MK2206 was injected intracerebroventricularly (i.c.v.). SAH grade, neurological scores, and brain water content were measured 24 h after SAH. Neuronal apoptosis was visualized by Fluoro-Jade C (FJC) staining. Western blotting was used to measure the levels of PERK, p-PERK, eIF2α, p-eIF2α, Akt, p-Akt, Bcl-2, Bax, and cleaved caspase-3. Our results showed that TMP effectively reduced neuronal apoptosis and improved neurobehavioral deficits 24 h after SAH. Administration of TMP reduced the abundance of p-PERK and p-eIF2α. In addition, TMP increased the p-Akt level and the Bcl-2/Bax ratio and decreased the level of cleaved caspase-3. The selective Akt inhibitor MK2206 abolished the anti-apoptotic effect of TMP at 24 h after SAH. Collectively, these results indicate that Akt-related anti-apoptosis through the PERK pathway is a major, potent mechanism of EBI. Further investigation of this pathway may provide a basis for the development of TMP as a clinical treatment.