Validation of organotypical hippocampal slice cultures as an ex vivo model of brain ischemia: different roles of NMDA receptors in cell death signalling after exposure to NMDA or oxygen and glucose deprivation

N-Methyl-D-aspartate receptors (NMDARs) are essential mediators of synaptic plasticity under normal physiological conditions. During brain ischemia, these receptors are excessively activated due to glutamate overflow and mediate excitotoxic cell death. Although organotypical hippocampal slice cultures are widely used to study brain ischemia in vitro by induction of oxygen and glucose deprivation (OGD), there is scant data regarding expression and functionality of NMDARs in such slice cultures. Here, we have evaluated the contribution of NMDARs in mediating excitotoxic cell death after exposure to NMDA or OGD in organotypical hippocampal slice cultures after 14 days in vitro (DIV14). We found that all NMDAR subunits were expressed at DIV14. The NMDARs were functional and contributed to cell death, as evidenced by use of the NMDAR antagonist MK-801 (dizocilpine). Excitotoxic cell death induced by NMDA could be fully antagonized by 10 μM MK-801, a dose that offered only partial protection against OGD-induced cell death. Very high concentrations of MK-801 (50–100 μM) were required to counteract cell death at long delays (48–72 h) after OGD. The relative high dose of MK-801 needed for long-term protection after OGD could not be attributed to down-regulation of NMDARs at the gene expression level. Our data indicate that NMDAR signaling is just one of several mechanisms underlying ischemic cell death and that prospective cytoprotective therapies must be directed to multiple targets.     

MK801对氧糖剥夺神经元P38MAPK和BCL2／BAX蛋白表达的影响

目的观察MK801的干预对氧糖剥夺神经元的P38 MAPK和bcl2／bax的影响,探讨其可能的作用途径。方法①原代培养的小鼠皮质神经元随机分成3组：对照组、氧糖剥夺组和氧糖剥夺MK801干预组。②MTT和流式细胞技术检测神经元的生存和凋亡情况。③western blot分别检测P-P38／P38和bcl2／bax的蛋白表达水平。结果①MK801的干预具有明显的神经元缺氧保护作用。②氧糖剥夺过程中,P-P38的表达上调,而bcl2降低,应用MK801干预后可有效逆转此种变化趋势。结论MK801可能经由P38 MAPK和bcl2途径发挥神经元缺氧的保护性作用。     

Tetramethylpyrazine Suppresses Transient Oxygen-Glucose Deprivation-Induced Connexin32 Expression and Cell Apoptosis via the ERK1/2 and p38 MAPK Pathway in Cultured Hippocampal Neurons

Tetramethylpyrazine (TMP) has been widely used in China as a drug for the treatment of various diseases. Recent studies have suggested that TMP has a protective effect on ischemic neuronal damage. However, the exact mechanism is still unclear. This study aims to investigate the mechanism of TMP mediated ischemic hippocampal neurons injury induced by oxygen-glucose deprivation (OGD). The effect of TMP on hippocampal neurons viability was detected by MTT assay, LDH release assay and apoptosis rate was measured by flow cytometry. TMP significantly suppressed neuron apoptosis in a concentration-dependent manner. TMP could significantly reduce the elevated levels of connexin32 (Cx32) induced by OGD. Knockdown of Cx32 by siRNA attenuated OGD injury. Moreover, our study showed that viability was increased in siRNA-Cx32-treated-neurons, and neuron apoptosis was suppressed by activating Bcl-2 expression and inhibiting Bax expression. Over expression of Cx32 could decrease neurons viability and increase LDH release. Furthermore, OGD increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the neuron injury and Cx32 up-regulation. Taken together, TMP can reverse the OGD-induced Cx32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathways.     

The role of β‐adrenergic receptors and p38MAPK signaling pathways in physiological processes of cardiosphere‐derived cells

The effects of β adrenergic receptors (β‐ARs) and p38 mitogen‐activated protein kinases (MAPK) pathways on cardiosphere‐derived cells (CDCs) are largely unknown. This study aimed to investigate the roles of β‐ARs and p38MAPK pathways on the proliferation, apoptosis, and differentiation capacity of CDCs. The CDCs were treated with β1‐AR blocker (Met group), β2‐AR antagonist (ICI group), and p38MAPK inhibitor (SB group), non‐selective β‐AR blocker (PRO group), and β‐AR agonist (ISO group). The viability, apoptotic rate and differentiation status of CDCs were determined by MST‐1 assay, flow cytometery, and Western blot, respectively. The CDCs viability significantly reduced in ICI group (all P < 0.05), and SB group had a significant high viability after 48 h treatment (P < 0.05). Compared with control group, all treated groups had a low apoptotic rate. After treatment for 72 h, ISO treatment elevated the expression of Nkx2.5, and could partially or fully attenuate the inhibitory effects of β‐AR antagonists and/or p38MAPK inhibitor. A similar overall trend of protein expression levels among all groups could be observed between protein pairs of cTnT and β1‐AR as well as c‐Kit and β2‐AR, respectively. These results suggested that β‐ARs and p38MAPK signaling pathways play crucial roles in the proliferation and differentiation of CDCs. Our findings should be helpful for better understanding the molecular mechanism underlying the physiological processes of CDCs.     

Beneficial Effect of Astragalosides on Stroke Condition Using PC12 Cells under Oxygen Glucose Deprivation and Reperfusion

Astragalosides (AST) are reported to be neuroprotective in focal cerebral ischemic models in vivo. In this study, the direct effect of AST against oxygen and glucose deprivation (OGD) including neuronal injury and the underlying mechanisms in vitro were investigated. 5 h OGD followed by 24 h of reperfusion [adding back oxygen and glucose (OGD-R)] was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 µg/mL) were added to the culture after 5 h of the OGD ischemic insult and was present during the reoxygenation phases. A key finding was that OGD-R decreased cell viability, increased lactate dehydrogenase, increased reactive oxygen species, apoptosis, autophagy, functional impairment of mitochondria, and endoplasmic reticulum stress in PC12 cells, all of which AST treatment significantly reduced. In addition, AST attenuated OGD-R-induced cell loss through P38 MAPK activation a neuroprotective effect blunted by SB203580, a specific inhibitor of P38 MAPK. Our data suggest that both apoptosis and autophagy are important characteristics of OGD-R-induced PC12 death and that treating PC12 cells with AST blocked OGD-R-induced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and endoplasmic reticulum stress. Our data provide identification of AST that can concomitantly inhibit multiple cells death pathways following OGD injuries in neural cells.     

在低氧预处理延迟心肌保护中钙网蛋白表达升高

本文分别在整体实验和细胞培养条件下研究钙网蛋白（calreticulin,CRT）在低氧预处理（hypoxic preconditioning,HPC）延迟心肌保护中的表达及其信号转导机制。（1）整体实验时Wistar大鼠随机分为3组：假手术（sham）组、仅结扎冠状动脉的心肌缺血（myocardial infarction,MI）组和HPC后再结扎冠状动脉的HPC＋MI组,分别于术后24h、14d和28d观察HPC对缺血后心功能和梗死区、危险区面积的影响;采用Western blot检测CRT表达以及p38丝裂素活化蛋白激酶（p38mitogenactivated protein kinase,p38MAPK）、应激活化蛋白激酶（stress-activated protein kinase,SAPK）活性。（2）原代培养Sprague—Dawley乳鼠心肌细胞,随机分为6组：低氧,复氧（hypoxia／reoxygenation,H／R）组、HPC组、HPC＋H,R组、p38MAPK抑制剂SB203580＋HPC＋H／R（SB＋HPC＋H／R）组、SAPK抑制剂SP600125＋HPC＋H瓜（SP＋HPC＋H／R）组和正常对照组;采用台盼蓝排斥实验、乳酸脱氢酶（1actate dehydrogenase,LDH）活性检测及流式细胞仪检测各组细胞损伤情况;采用Western blot检测CRT表达及p38MAPK、SAPK的磷酸化水平。主要结果如下：（1）整体动物实验结果表明,HPC改善缺血对心肌左室压力最大上升,下降速度（＋dp／dtmax）的抑制,限制心肌梗死面积;HPC后CRT表达呈动态变化：术后24h时HPC＋MI组CRT表达增高106％（P〈0．05vsMI组）,以危险区最为显著;14d至28d表达逐步降低。相关分析显示,术后24h时CRT表达量与心功能呈正相关（r=0．9867,P〈0．05）,与梗死面积呈负相关（r=-0．9709,P〈0．05）。（2）细胞培养实验结果表明,HPC可减轻H／R诱导的心肌细胞LDH漏出,增加心肌细胞存活率,降低细胞凋亡;单纯HPC可诱导CRT表达轻度增加（222％,P〈0．05vs对照组）,而损伤性H／R诱导CRT过表达（503％,P〈0．05vs对照组）,HPC可降低H／R诱导CRT表达升高的幅度;p38MAPK活性与HPC诱导的CRT表达呈正相关（r=0．9021,P〈0．05）,而SAPK活性与其呈负相关（r=-0．8211,P〈0．05）。由此得出结论：（1）整体实验中HPC可明显改善缺血后心脏的收缩与舒张功能,限制心肌梗死范围,促进危险区心肌恢复;心肌梗死早期,HPC诱导CRT表达上调,参与心肌保护;（2）细胞培养实验中HPC可诱导CRT适度表达,增强原代培养乳鼠心肌细胞对H／R损伤的抵抗力;p38MAPK可能介导HPC诱导的CRT表达,而SAPK激活可能不利于心肌保护。     

An inhibitor of p38 mitogen-activated protein kinase protects neonatal cardiac myocytes from ischemia

Cellular ischemia results in activation of a number of kinases, including p38 mitogen-activated protein kinase (MAPK); however, it is not yet clear whether p38 MAPK activation plays a role in cellular damage or is part of a protective response against ischemia. We have developed a model to study ischemia in cultured neonatal rat cardiac myocytes. In this model, two distinct phases of p38 MAPK activation were observed during ischemia. The first phase began within 10 min and lasted less than 1 h, and the second began after 2 h and lasted throughout the ischemic period. Similar to previous studies using in vivo models, the nonspecific activator of p38 MAPK and c-Jun NH2-terminal kinase, anisomycin, protected cardiac myocytes from ischemic injury, decreasing the release of cytosolic lactate dehydrogenase by approximately 25%. We demonstrated, however, that a selective inhibitor of p38 MAPK, SB 203580, also protected cardiac myocytes against extended ischemia in a dose-dependent manner. The protective effect was seen even when the inhibitor was present during only the second, sustained phase of p38 MAPK activation. We found that ischemia induced apoptosis in neonatal rat cardiac myocytes and that SB 203580 reduced activation of caspase-3, a key event in apoptosis. These results suggest that p38 MAPK induces apoptosis during ischemia in cardiac myocytes and that selective inhibition of p38 MAPK could be developed as a potential therapy for ischemic heart disease.     

Chloride channel inhibition prevents ROS-dependent apoptosis induced by ischemia-reperfusion in mouse cardiomyocytes.

Apoptosis of cardiomyocytes following ischemia and Apoptosis of cardiomyocytes following ischemia and known about the mechanism by which it is induced. Recently, essential roles of a Cl- channel whose activity triggers the apoptotic volume decrease and of reactive oxygen species (ROS) in activation of this channel have been identified in mitochondrion-mediated apoptosis. Therefore, in this study, involvement of Cl- channels and ROS in apoptosis was studied in primary mouse cardiomyocyte cultures subjected to ischemia-reperfusion. Apoptotic cell death as measured by caspase-3 activation, chromatin condensation, DNA laddering, and cell viability reduction was observed tens of hours after reperfusion but never immediately after ischemia. A non-selective Cl-channel blocker (DIDS or NPPB) rescued cells from apoptotic death when applied during the reperfusion, but not ischemia, period. Another blocker relatively specific to the volume-sensitive outwardly rectifying (VSOR) Cl-channel (phloretin) was also effective in protecting ischemic cardiomyocytes from apoptosis induced by reperfusion. A profound increase in intracellular ROS was detected in cardiomyocytes during the reperfusion, but not ischemia, period. Scavengers for ROS, H2O2 and superoxide all inhibited apoptosis induced by ischemia-reperfusion. Thus, it is concluded that the mechanism by which cardiomyocyte apoptosis is induced by ischemia-reperfusion involves VSOR Cl- channel activity and intracellular ROS production.     

Expression of NMDA Receptor and its Effect on Cell Proliferation in the Subventricular Zone of Neonatal Rat Brain

We investigated the involvement of N-methyl d-aspartate receptor (NMDAR) in neurogenesis of rat’s subventricular zone (SVZ). For this purpose, we determined expression of the NMDAR subunits NR1, NR2A, and NR2B in SVZ of the neonatal Sprague-Dawley rats using immunohistochemical techniques. All three NMDAR subunits were expressed during postnatal day (PND)-1 to PND-28 whereas each subunit showed a distinct expression pattern. We also examined the functional effect of this receptor on cell proliferation in this region and, in this regard, the animals received either intraperitoneal injection of NMDAR agonist NMDA (2 mg/kg/day) or selective non-competitive NMDAR antagonist MK-801 (10 mg/kg) or NR2B antagonist Ro25-6981 (40 mg/kg), respectively, at PND-3. A significant developmental increase of the total cell density was observed at PND-7 (P < 0.05) while proliferating cell nuclear antigen-positive cell density was significantly increased at PND-14 (P < 0.05) and at PND-28 (P < 0.05) in the SVZ after NMDA (2 mg/kg/day) injection. Our data show that the NMDAR activation promoted the cell proliferation in SVZ during the neonatal period. We, therefore, inferred that NMDAR is expressed in SVZ of the neonatal rat brain and can promote neurogenesis, as through cell proliferation process in that region, and can thus be used as a potential therapeutic target in neurodegenerative diseases.     

The role of differential activation of p38-mitogen-activated protein kinase in preconditioned ventricular myocytes.

Activation of protein kinase C (PKC) and more recently mitogen-activated protein kinases (MAPKs) have been associated with the cardioprotective effect of ischemic preconditioning. We examined the interplay between these kinases in a characterized model of ischemic preconditioning in cultured rat neonatal ventricular cardiocytes where ectopic expression of active PKC-delta results in protection. Two members of the MAPK family, p38 and p42/44, were activated transiently during preconditioning by brief simulated ischemia/reoxygenation. Overexpression of active PKC-delta, rather than augmenting, completely abolished this activation. We therefore determined whether a similar process occurred during lethal prolonged simulated ischemia. In contrast to ischemia, brief, lethal-simulated ischemia activated only p38 (2.8+/-0.45 vs. basal, P<0.01), which was attenuated by expression of active PKC-delta or by preconditioning (0.48+/-0.1 vs. ischemia, P<0.01). To determine whether reduced p38 activation was the cause or an effect of protection, we used SB203580, a p38 inhibitor. SB203580 reduced ischemic injury (CK release 38.0+/-3.1%, LDH release 77.3+/-4.0%, and MTT bioreduction 127.1+/-4.8% of control, n=20, P<0.05). To determine whether p38 activation was isoform selective, myocytes were infected with adenoviruses encoding wild-type p38alpha or p38beta. Transfected p38alpha and beta show differential activation (P<0.001) during sustained simulated ischemia, with p38alpha remaining activated (1.48+/-0.36 vs. basal) but p38beta deactivated (0.36+/-0.1 vs. basal, P<0.01). Prior preconditioning prevented the activation of p38alpha (0.65+/-0.11 vs. ischemia, P<0.05). Moreover, cells expressing a dominant negative p38alpha, which prevented ischemic p38 activation, were resistant to lethal simulated ischemia (CK release 82.9+/-3.9% and MTT bioreduction 130.2+/-6.5% of control, n=8, P<0.05). Thus, inhibition of p38alpha activation during ischemia reduces injury and may contribute to preconditioning-induced cardioprotection in this model.     

Pituitary adenylate cyclase-activating polypeptide is up-regulated in cortical pyramidal cells after focal ischemia and protects neurons from mild hypoxic/ischemic damage

The protective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in stroke models is poorly understood. We studied patterns of PACAP, vasoactive intestinal peptide, and the PACAP-selective receptor PAC1 after middle cerebral artery occlusion and neuroprotection by PACAP in cortical cultures exposed to oxygen/glucose deprivation (OGD). Within hours, focal ischemia caused a massive, NMDA receptor (NMDAR)-dependent up-regulation of PACAP in cortical pyramidal cells. PACAP expression dropped below the control level after 2 days and was normalized after 4 days. Vasoactive intestinal peptide expression was regulated oppositely to that of PACAP. PAC1 mRNA showed ubiquitous expression in neurons and astrocytes with minor changes after ischemia. In cultured cortical neurons PACAP27 strongly activated Erk1/2 at low and p38 MAP kinase at higher nanomolar concentrations via PAC1. In astrocyte cultures, effects of PACAP27 on Erk1/2 and p38 were weak. During OGD, neurons showed severely reduced Erk1/2 activity and dephosphorylation of Erk1/2-regulated Ser112 of pro-apoptotic Bad. PACAP27 stimulation counteracted Erk1/2 inactivation and Bad dephosphorylation during short-term OGD but was ineffective after expanded OGD. Consistently, PACAP27 caused MEK-dependent neuroprotection during mild but not severe hypoxic/ischemic stress. While PACAP27 protected neurons at 1–5 nmol/L, full PAC1 activation by 100 nmol/L PACAP exaggerated hypoxic/ischemic damage. PACAP27 stimulation of astrocytes increased the production of Akt-activating factors and conferred ischemic tolerance to neurons. Thus, ischemia-induced PACAP may act via neuronal and astroglial PAC1. PACAP confers protection to ischemic neurons by maintaining Erk1/2 signaling via neuronal PAC1 and by increasing neuroprotective factor production via astroglial PAC1.     

miR-20b-5p靶向MAPK1调控脑出血过程中脑微血管内皮细胞铁死亡

摘要 目的：探讨miR-20b-5p对氧糖剥夺（OGD）/Hemin处理的脑微血管内皮细胞（BMVEC）功能的影响及机制。方法：将BMVEC分为Control组、agomir-NC组、agomir-miR-20b-5p组、antagomir-NC组和antagomir-miR-20b-5p组。使用Lipofectamine 2000试剂对细胞进行相应的转染处理。BMVEC转染后，将BMVEC再分为Control组、OGD/Hemin组（O/H组）、OGD/Hemin+agomir-NC组（O/H+agomir-NC组）、OGD/Hemin+agomir-miR-20b-5p组（O/H+agomir-miR-20b-5p组）、OGD/Hemin+antagomir-NC组（O/H+antagomir-NC组）和OGD/Hemin+antagomir-miR-20b-5p组（O/H+antagomir-miR-20b-5p组）。Control组BMVEC正常培养，其他组BMVEC进行OGD/Hemin处理。MTT法检测BMVEC增殖，TUNEL染色检测BMVEC凋亡，Transwell检测BMVEC迁移。使用试剂盒检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和丙二醛（MDA）水平。使用Iron Assay试剂盒检测Fe²⁺含量。通过qRT-PCR检测miR-20b-5p和MAPK1 mRNA水平。通过Western blot检测MAPK1、Bax、Bcl-2、谷胱甘肽过氧化物酶4（GPX4）和前列腺素内过氧化物合酶2（PTGS2）蛋白表达水平。通过免疫荧光染色检测MAPK1的荧光强度水平。结果：与Control组和agomir-NC组比较，agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高（P<0.05）。与Control组和antagomir-NC组比较，antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低（P<0.05）。与Control组比较，O/H组BMVEC中的miR-20b-5p水平降低，细胞活力降低，TUNEL阳性率和Bax蛋白表达水平升高，Bcl-2蛋白表达水平降低，迁移数量降低，SOD和GSH-Px活性降低，MDA含量升高，Fe²⁺含量和PTGS2的蛋白表达水平升高，GPX4的蛋白表达水平降低，MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高（P<0.05）。与O/H组和O/H+agomir-NC组比较，O/H+agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高，细胞活力升高，TUNEL阳性率和Bax蛋白表达水平降低，Bcl-2蛋白表达水平升高，迁移数量升高，SOD和GSH-Px活性升高，MDA含量降低，Fe²⁺含量和PTGS2的蛋白表达水平降低，GPX4的蛋白表达水平升高，MAPK1的mRNA和蛋白表达水平以及相对荧光强度降低（P<0.05）。与O/H组和O/H+antagomir-NC组比较，O/H+antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低，细胞活力降低，TUNEL阳性率和Bax蛋白表达水平升高，Bcl-2蛋白表达水平降低，迁移数量降低，SOD和GSH-Px活性降低，MDA含量升高，Fe²⁺含量和PTGS2的蛋白表达水平升高，GPX4的蛋白表达水平降低，MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高（P<0.05）。结论：本研究表明上调miR-20b-5p通过抑制OGD/Hemin处理的BMVEC中MAPK1的表达从而抑制了铁死亡途径。     

Interleukin-17A-mediated alleviation of cortical astrocyte ischemic injuries affected the neurological outcome of mice with ischemic stroke

We previously reported that astrocytes are the main sources of interleukin (IL)-17A production that could aggravate neuronal injuries in ischemic stroke. However, the effects of IL-17A on ischemic astrocytes themselves and the underlying molecular mechanism are still unclear. In this study, we found that recombinant mouse (rm) IL-17A could significantly (P < 0.05 or <0.001) alleviate 1-hour oxygen-glucose deprivation (OGD)/reoxygenation (R) 24-hour–induced ischemic injuries in cortical astrocytes with a dose-dependent manner (n = 6 per group). The Western blot and cell cycle analysis results revealed that rmIL-17A significantly ( P < 0.05) inhibited procaspase-3 cleavage without affecting cell proliferation in 1-hour OGD/R 24-hour–treated cortical astrocytes (n = 6 per group). Among the five IL-17 receptor subunits (IL-RA, -RB, -RC, -RD, and -RE), only IL-17RA ( P < 0.01) and -17RC ( P < 0.05) membrane translocation (not messenger RNA and protein) levels were downregulated in cortical astrocytes following 1-hour OGD/reperfusion 24 hours, and rmIL-17A could significantly ( P < 0.05 or <0.001) inhibit this downregulation (n = 6 per group). To further verify the impact of IL-17A on the neurological outcome of ischemic stroke, we found that the intracerebroventricular injection of IL-17A neutralizing monoclonal antibody remarkably ( P < 0.001) reduced the astrocyte activation and improve neurological function ( P < 0.05 or <0.01) of mice following 1-hour middle cerebral artery occlusion/reperfusion (R) 3 to 7 days (n = 6 or 8 per group). These results suggested that IL-17A-mediated alleviation of cortical astrocyte ischemic injuries could affect the neurological outcome of mice with ischemic stroke, which might be mainly dependent on the cell apoptosis pathway through inhibiting the downregulation of IL-17RA and -17RC membrane translocations.     

Calreticulin induces delayed cardioprotection through mitogen-activated protein kinases

Hypoxic preconditioning (HPC) attenuates tissue injury caused by ischemia/reperfusion. The protective mechanisms of HPC involve up-regulation of the protective proteins and mitigation of cellular calcium overload. Calreticulin (CRT), a Ca(2+)-binding chaperone, plays an important role in regulating calcium homeostasis and folding of proteins. The role of CRT in cardioprotection of HPC and the pathways determining CRT expression during HPC are not clear. In this work, 2-DE and MALDI-MS were employed to analyze CRT differential expression in cardiomyocytes subjected to transient hypoxia. Western blotting analysis was used to detect the CRT expression and activities of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH(2)-terminal kinase (JNK) in myocardium subjected to ischemia with and without HPC and sham operation. The hearts from HPC group were more resistant to sustained ischemia and had much stronger phosphorylation of p38 MAPK, with a reduced phosphorylation of JNK, than controls. The CRT expression was positively correlated with the phosphorylation of p38 MAPK and negatively correlated with the level of JNK phosphorylation. Furthermore, inhibition of the p38 MAPK with SB202190 abolished, while inhibition of the JNK with SP600125 enhanced the CRT up-regulation in cardiomyocytes induced by HPC. The results indicate that HPC up-regulates CRT expression through the MAPK signaling pathways.     

The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells

Recent reports suggest that N-methyl-d-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination.     

Non‐hemolytic enterotoxin of Bacillus cereus induces apoptosis in Vero cells

Bacillus cereus is an opportunistic pathogen that often causes foodborne infectious diseases and food poisoning. Non‐hemolytic enterotoxin (Nhe) is the major toxin found in almost all enteropathogenic B. cereus and B. thuringiensis isolates. However, little is known about the cellular response after Nhe triggered pore formation on cell membrane. Here, we demonstrate that Nhe induced cell cycle arrest at G₀/G₁ phase and provoked apoptosis in Vero cells, most likely associated with mitogen‐activated protein kinase (MAPK) and death receptor pathways. The influx of extracellular calcium ions and increased level of reactive oxygen species in cytoplasm were sensed by apoptosis signal‐regulating kinase 1 (ASK1) and p38 MAPK. Extrinsic death receptor Fas could also promote the activation of p38 MAPK. Subsequently, ASK1 and p38 MAPK triggered downstream caspase‐8 and 3 to initiate apoptosis. Our results clearly demonstrate that ASK1, and Fas‐p38 MAPK‐mediated caspase‐8 dependent pathways are involved in apoptotic cell death provoked by the pore‐forming enterotoxin Nhe.     

Phytic acid exerts protective effects in cerebral ischemia-reperfusion injury by activating the anti-oxidative protein sestrin2

ABSTRACT

Cerebral ischemia reperfusion (I/R) is a therapeutic strategy for ischemia; however, it usually causes injury by the aspect of inflammation and neuron apoptosis. This investigation aims to investigate the protective effects of phytic acid (IP6) for cerebral I/R injury in vitro. PC-12 cells under Oxygen and glucose deprivation/reperfusion (OGD/R) were performed to mimic cerebral I/R. IP6 was pretreated before PC-12 cells under OGD/R treatment. The data showed that IP6 activated the expression of sestrin2 in OGD/R injured PC-12 cells. IP6 inhibited OGD/R induced inflammation, oxidative stress, and apoptosis by activating sestrin2. Besides, p38 MAPK may mediate the effects of sestrin2 activated by IP6. Therefore, IP6 can be a potential drug to prevent neurological damage in cerebral I/R injury.     

Effect of miR-499a-5p on damage of cardiomyocyte induced by hypoxia-reoxygenation via downregulating CD38 protein

The aim is to investigate the mechanism of miR-499a-5p on the damage of cardiomyocyte induced by hypoxia/reoxygenation. The activity of lactate dehydrogenase (LDH), apoptosis rate and the expression of miR-499a-5p and cluster of differentiation 38 (CD38) in hypoxia-reoxygenation model cells were detected by LDH Cytotoxicity Assay Kit, flow cytometry, real-time polymerase chain reaction, and Western blot analysis, respectively. Apoptosis, the activity of LDH was detected after overexpression of miR-499a-5p or silencing of CD38 in H9c2 cells. The target relationship between miR-499a-5p and CD38 was verified by Targetscan online prediction and dual-luciferase assay. Apoptosis, the activity of LDH was detected after overexpression of miR-499a-5p and CD38. Apoptosis, the activity of LDH and the expression of CD38 were increased (P < .05) while expression of miR-499a-5p was decreased (P < .05) in hypoxia/reoxygenation model cells. Apoptosis and the activity of LDH in H9c2 cells after overexpression of miR-499a-5p or silence of CD38 were decreased (P < .05). The results of Targetscan online prediction and dual-luciferase assay indicated that CD38 was a potential target gene of miR-499a-5p. Overexpression of CD38 could reverse the inhibition of miR-499a-5p on LDH activity and apoptosis in H9c2 cells. miR-499a-5p could relief the injury of cardiomyocytes induced by hypoxia/reoxygenation via targeting CD38.     

Denitrosylation of S-nitrosylated OGT is triggered in LPS-stimulated innate immune response

Rad is a member of a subclass of small GTP-binding proteins, the RGK family. In the present study we investigated the role of Rad protein in regulating cardiomyocyte viability. DNA fragmentation and TUNEL assays demonstrated that Rad promoted rat neonatal cardiomyocyte apoptosis. Rad silencing fully blocked serum deprivation induced apoptosis, indicating Rad is necessary for trigger cardiomyocyte apoptosis. Rad overexpression caused a dramatic decrease of the anti-apoptotic molecule Bcl-x_L, whereas Bcl-x_L overexpression protected cardiomyocytes against Rad-induced apoptosis. Rad-triggered apoptosis was mediated by the activation of p38 MAPK. The p38 blocker SB203580 effectively protected cardiomyocytes against Rad-evoked apoptosis.