Long noncoding NONRATT021972 siRNA normalized abnormal sympathetic activity mediated by the upregulation of P2X<Subscript>7</Subscript> receptor in superior cervical ganglia after myocardial ischemia

Previous studies showed that the upregulation of the P2X₇ receptor in cervical sympathetic ganglia was involved in myocardial ischemic (MI) injury. The dysregulated expression of long noncoding RNAs (lncRNAs) participates in the onset and progression of many pathological conditions. The aim of this study was to investigate the effects of a small interfering RNA (siRNA) against the NONRATT021972 lncRNA on the abnormal changes of cardiac function mediated by the up-regulation of the P2X₇ receptor in the superior cervical ganglia (SCG) after myocardial ischemia. When the MI rats were treated with NONRATT021972 siRNA, their increased systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), low-frequency (LF) power, and LF/HF ratio were reduced to normal levels. However, the decreased high-frequency (HF) power was increased. GAP43 and tyrosine hydroxylase (TH) are markers of nerve sprouting and sympathetic nerve fibers, respectively. We found that the TH/GAP43 value was significantly increased in the MI group. However, it was reduced after the MI rats were treated with NONRATT021972 siRNA. The serum norepinephrine (NE) and epinephrine (EPI) concentrations were decreased in the MI rats that were treated with NONRATT021972 siRNA. Meanwhile, the increased P2X₇ mRNA and protein levels and the increased p-ERK1/2 expression in the SCG were also reduced. NONRATT021972 siRNA treatment inhibited the P2X₇ agonist BzATP-activated currents in HEK293 cells transfected with pEGFP-P2X₇. Our findings suggest that NONRATT021972 siRNA could decrease the upregulation of the P2X₇ receptor and improve the abnormal changes in cardiac function after myocardial ischemia.     

LncRNA NONRATT021972 involved the pathophysiologic processes mediated by P2X<Subscript>7</Subscript> receptors in stellate ganglia after myocardial ischemic injury

Adenosine triphosphate (ATP) acts on P2X receptors to initiate signal transmission. P2X₇ receptors play a role in the pathophysiological process of myocardial ischemic injury. Long noncoding RNAs (lncRNAs) participate in numerous biological functions independent of protein translation. LncRNAs are implicated in nervous system diseases. This study investigated the effects of NONRATT021972 small interference RNA (siRNA) on the pathophysiologic processes mediated by P2X₇ receptors in stellate ganglia (SG) after myocardial ischemic injury. Our results demonstrated that the expression of NONRATT021972 in SG was significantly higher in the myocardial ischemic (MI) group than in the control group. Treatment of MI rats with NONRATT021972 siRNA, the P2X₇ antagonist brilliant blue G (BBG), or P2X₇ siRNA improved the histology of injured ischemic cardiac tissues and decreased the elevated concentrations of serum myocardial enzymes, creatine kinase (CK), CK isoform MB (CK-MB), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) compared to the MI rats. NONRATT021972 siRNA, BBG, or P2X₇ siRNA treatment in MI rats decreased the expression levels of P2X₇ immunoreactivity, P2X₇ messenger RNA (mRNA), and P2X₇ protein, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) in the SG compared to MI rats. NONRATT021972 siRNA treatment prevented the pathophysiologic processes mediated by P2X₇ receptors in the SG after myocardial ischemic injury.     

LncRNA NONRATT021972 siRNA attenuates P2X7 receptor expression and inflammatory cytokine production induced by combined high glucose and free fatty acids in PC12 cells

Diabetic neuropathy (DNP) is a frequent chronic complication of diabetes mellitus with potentially life-threatening outcomes. High glucose and elevated free fatty acids (FFAs) have been recently recognized as major causes of nervous system damage in diabetes. Our previous study has indicated extracellular stimuli, such as high glucose and/or FFA stress, may activate the p38 mitogen-activated protein kinase (MAPK) signaling pathway and induce a p38 MAPK-dependent sensitization of the P2X7 receptor and release of inflammatory factors in PC12 cells, while the mechanisms underlying remain to be elucidated. Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes, including activation of a series of pathway signalings. Here, we showed combined high d-glucose and FFAs (HGHF) induced an increment of lncRNA-NONRATT021972 (NONCODE ID, nc021972) in PC12 cells. Nc021972 small interference RNA (siRNA) alleviated HGHF-induced activation of p38 MAPK, expression of the P2X7 receptor, and [Ca²⁺]_i increment upon P2X7 receptor activation. Further experiments showed that there existed a crosstalk between nc021972 and the p38 MAPK signaling pathway. Inhibition of p38 MAPK signaling decreased nc021972-induced expression of the P2X7 receptor and [Ca²⁺]_i increment upon P2X7 receptor activation. Also, nc021972 siRNA inhibited HGHF-induced PC12 release of TNF-α and IL-6 and rescued decreased cell viability mediated by the P2X7 receptor. Therefore, inhibition of nc021972 may serve as a novel therapeutic strategy for diabetes complicated with nervous inflammatory diseases.     

Change of Beclin‐1 dependent on ATP, [Ca2+]i and MMP in PC12 cells following oxygen‐glucose deprivation‐reoxygenation injury

Autophagy is usually up‐regulated to provide more ATP in response to starvation or OGD (oxygen‐glucose deprivation), but the relationship between autophagy and ATP, [Ca²⁺]_i (intracellular free Ca²⁺ concentration) or MMP (mitochondrial membrane potential) during reoxygenation is not yet fully clear. The role of autophagy is unknown in PC12 cells subjected to 2 h OGD with different time points of reoxygenation. In the present study, we showed that Beclin‐1 was up‐regulated beginning at 0 h reoxygenation peaking at 24 h and lasting for 48 h. Cell viability was decreased from 0 to 48 h reoxygenation, reaching its minimum at 10 h reoxygenation. ATP was decreased from 0 to 10 h reoxygenation, reaching its minimum at 4 h reoxygenation. A significant negative correlation was observed between ATP and Beclin‐1 (r = ?0.61, P<0.05) at 0 h reoxygenation, but ATP was not significant related (r = 0.24, P>0.05) to Beclin‐1 at 24 h reoxygenation. Besides, Nimodipine, a calcium antagonist, significantly reduced [Ca²⁺]_i and Beclin‐1, but increased MMP in OGD/R‐treated cells. At 24 h reoxygenation, Beclin‐1 expression reached its maximum, cell viability continued to increase, and ATP was higher than that before OGD. These results suggest that energy metabolism dysfunction can induce autophagy during OGD in PC12 cells. Increased [Ca²⁺]_i and decreased MMP may induce autophagy during reoxygenation in PC12 cells. Autophagy may be a protective effect on PC12 cells treated with different time points of reoxygenation after 2 h OGD.     

Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells

Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X₇ receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined. We characterized the role of P2X₇ receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X₇ receptors. Functional inhibition of P2X₇ receptors by P2X₇ receptor selective antagonists, 5′-triphosphate, periodate-oxidized 2′,3′-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X₇ receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were decreased in either RA- or oATP-differentiated or P2X₇ receptor knockdown N2a cells. Simply cultured N2a cells in low Ca²⁺ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X₇ receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X₇ receptors are involved in neuronal differentiation. We provide additional evidence shown that the ATP release-activated P2X₇ receptor is important in maintaining cell survival of N2a neuroblastoma cells.     

P2X<Subscript>7</Subscript>R modulation of visually evoked synaptic responses in the retina

P2X₇Rs are distributed throughout all layers of the retina, and thus, their localisation on various cell types puts into question their specific site(s) of action. Using a dark-adapted, ex vivo mouse retinal whole mount preparation, the present study aimed to characterise the effect of P2X₇R activation on light-evoked, excitatory RGC ON-field excitatory post-synaptic potentials (fEPSPs) and on outer retinal electroretinogram (ERG) responses under comparable conditions. The pharmacologically isolated NMDA receptor-mediated RGC ON-fEPSP was reduced in the presence of BzATP, an effect which was significantly attenuated by A438079 and other selective P2X₇R antagonists A804598 or AF27139. In physiological Krebs medium, BzATP induced a significant potentiation of the ERG a-wave, with a concomitant reduction in the b-wave and the power of the oscillatory potentials. Conversely, in the pharmacologically modified Mg²⁺-free perfusate, BzATP reduced both the a-wave and b-wave. The effects of BzATP on the ERG components were suppressed by A438079. A role for P2X₇R function in visual processing in both the inner and outer retina under physiological conditions remains controversial. The ON-fEPSP was significantly reduced in the presence of A804598 but not by A438079 or AF27139. Furthermore, A438079 did not have any effect on the ERG components in physiological Krebs but potentiated and reduced the a-wave and b-wave, respectively, when applied to the pharmacologically modified medium. Therefore, activation of P2X₇Rs affects the function in the retinal ON pathway. The presence of a high concentration of extracellular ATP would most likely contribute to the modulation of visual transmission in the retina in the pathophysiological microenvironment.     

P2X<Subscript>3</Subscript> Receptor Involvement in Pain States

The understanding of how pain is processed at each stage in the peripheral and central nervous system is the precondition to develop new therapies for the selective treatment of pain. In the periphery, ATP can be released from various cells as a consequence of tissue injury or visceral distension and may stimulate the local nociceptors. The highly selective distribution of P2X₃ and P2X_2/3 receptors within the nociceptive system has inspired a variety of approaches to elucidate the potential role of ATP as a pain mediator. Depolarization by ATP of neurons in pain–relevant neuronal structures such as trigeminal ganglion, dorsal root ganglion, and spinal cord dorsal horn neurons are well investigated. P2X receptor-mediated afferent activation appears to have been implicated in visceral and neuropathic pain and even in migraine and cancer pain. This article reviews recently published research describing the role that ATP and P2X receptors may play in pain perception, highlighting the importance of the P2X₃ receptor in different states of pain.     

Involvement of P2X and P2Y receptors in microglial activation in vivo

Microglial cells are the primary immune effector cells in the brain. Extracellular ATP, e.g., released after brain injury, may initiate microglial activation via stimulation of purinergic receptors. In the rat nucleus accumbens (NAc), the involvement of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated. A stab wound in the NAc increased immunoreactivity (IR) for P2X_1,2,4,7 and P2Y_1,2,4,6,12 receptors on microglial cells when visualized with confocal laser scanning microscopy. A prominent immunolabeling of P2X₇ receptors with antibodies directed against the ecto- or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells. Additionally, the P2X₇ receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt. Four days after local application of the agonists α,βmeATP, ADPβS, 2MeSATP, and BzATP, an increase in OX 42- and G. simplicifolia isolectin-IR was observed around the stab wound, quantified both densitometrically and by counting the number of ramified and activated microglial cells, whereas UTPγS appeared to be ineffective. The P2 receptor antagonists PPADS and BBG decreased the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects. Further, the intra-accumbally applied P2X₇ receptor agonist BzATP induced an increase in the number of caspase-3-positive cells. These results indicate that ATP, acting via different P2X and P2Y receptors, is a signaling molecule in microglial cell activation after injury in vivo. The up-regulation of P2X₇-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects.     

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X<Subscript>7</Subscript>-KO mice

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X₇-invalidated (KO) mice was tested. Low concentrations (1–100 μM) of ATP transiently increased the intracellular concentration of calcium ([Ca²⁺]_i) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X₇ receptors. One millimolar ATP provoked a sustained increase in the [Ca²⁺]_i only in WT mice. The response to 10 μM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K⁺]_i) and stimulated the secretion of interleukin-1β (IL-1β) only in cells from WT mice. Ten micromolar ATP in combination with 3 μM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1β was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 μM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 μM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 μM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X₄ receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K⁺]_i triggered the secretion of IL-1β. Pore formation was also triggered by activation of P2X₄ receptors. Higher concentrations of ATP elicited similar responses after binding to P2X₇ receptors. The expression of the P2X₇ receptors was also coupled to a better response to P2Y receptors.     

P2X7 nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons

Apoptosis is a major cause of cell death in the nervous system. It plays a role in embryonic and early postnatal brain development and contributes to the pathology of neurodegenerative diseases. Here, we report that activation of the P2X₇ nucleotide receptor (P2X₇R) in rat primary cortical neurons (rPCNs) causes biochemical (i.e., caspase activation) and morphological (i.e., nuclear condensation and DNA fragmentation) changes characteristic of apoptotic cell death. Caspase-3 activation and DNA fragmentation in rPCNs induced by the P2X₇R agonist BzATP were inhibited by the P2X₇R antagonist oxidized ATP (oATP) or by pre-treatment of cells with P2X₇R antisense oligonucleotide indicating a direct involvement of the P2X₇R in nucleotide-induced neuronal cell death. Moreover, Z-DEVD-FMK, a specific and irreversible cell permeable inhibitor of caspase-3, prevented BzATP-induced apoptosis in rPCNs. In addition, a specific caspase-8 inhibitor, Ac-IETD-CHO, significantly attenuated BzATP-induced caspase-9 and caspase-3 activation, suggesting that P2X₇R-mediated apoptosis in rPCNs occurs primarily through an intrinsic caspase-8/9/3 activation pathway. BzATP also induced the activation of C-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated kinases (ERK1/2) in rPCNs, and pharmacological inhibition of either JNK1 or ERK1/2 significantly reduced caspase activation by BzATP. Taken together, these data indicate that extracellular nucleotides mediate neuronal apoptosis through activation of P2X₇Rs and their downstream signaling pathways involving JNK1, ERK and caspases 8/9/3.     

Propofol Protects Against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Oxidative Injury in Differentiated PC12 Cells via Inhibition of Ca<Superscript>2+</Superscript>-Dependent NADPH Oxidase

Propofol (2,6-diisopropylphenol) is a widely used general anesthetic with anti-oxidant activities. This study aims to investigate protective capacity of propofol against hydrogen peroxide (H₂O₂)-induced oxidative injury in neural cells and whether the anti-oxidative effects of propofol occur through a mechanism involving the modulation of NADPH oxidase (NOX) in a manner of calcium-dependent. The rat differentiated PC12 cell was subjected to H₂O₂ exposure for 24 h to mimic a neuronal in vitro model of oxidative injury. Our data demonstrated that pretreatment of PC12 cells with propofol significantly reversed the H₂O₂-induced decrease in cell viability, prevented H₂O₂-induced morphological changes, and reduced the ratio of apoptotic cells. We further found that propofol attenuated the accumulation of malondialdehyde (biomarker of oxidative stress), counteracted the overexpression of NOX core subunit gp91^phox (NOX2) as well as the NOX activity following H₂O₂ exposure in PC12 cells. In addition, blocking of L-type Ca²⁺ channels with nimodipine reduced H₂O₂-induced overexpression of NOX2 and caspase-3 activation in PC12 cells. Moreover, NOX inhibitor apocynin alone or plus propofol neither induces a significant downregulation of NOX activity nor increases cell viability compared with propofol alone in the PC12 cells exposed to H₂O₂. These results demonstrate that the protective effects of propofol against oxidative injury in PC12 cells are mediated, at least in part, through inhibition of Ca²⁺-dependent NADPH oxidase.     

Differential Regulation of Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Signaling and Membrane Trafficking by Multiple P2 Receptors in Brown Adipocytes

Extracellular ATP triggers changes in intracellular Ca²⁺, ion channel function, and membrane trafficking in adipocytes. The aim of the present study was to determine which P2 receptors might mediate the Ca²⁺ signaling and membrane trafficking responses to ATP in brown fat cells. RT-PCR was used to determine which P2 receptors are expressed in brown fat cells. Responses to nucleotide agonists and antagonists were characterized using fura-2 fluorescence imaging of Ca²⁺ responses, and FM 1-43 fluorescence imaging and membrane capacitance measurements to assess membrane trafficking. The pharmacology of the Ca²⁺ responses fits the properties of the P2Y receptors for which mRNA is expressed, but the agonist and antagonist sensitivity of the membrane-trafficking response was not consistent with any P2 receptor described to date. Brown adipocytes expressed mRNA for P2Y₂, P2Y₆, and P2Y₁₂ metabotropic receptors and P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, and P2X₇ ionotropic receptors. The agonists ATP, ADP, UTP, UDP and 2′, 3′-(benzoylbenzoyl) ATP (BzATP) increased intracellular Ca²⁺, while 100 μM suramin, pyridoxal-phosphate-6-azophenyl-2′ 4′-disulfonic acid (PPADS), or Reactive Blue 2 partially blocked Ca²⁺ responses. ATP, but not ADP, UTP, UDP or BzATP activated membrane trafficking. The membrane response could be blocked completely with 1 μM PPADS but not by the antagonist MRS2179. We conclude that multiple P2 receptors mediate the ATP responses of brown fat cells, and that membrane trafficking is regulated by a P2 receptor showing unusual properties.     

Adenosine‐5′‐triphosphate suppresses proliferation and migration capacity of human endometrial stem cells

Extracellular ATP through the activation of the P2X and P2Y purinergic receptors affects the migration, proliferation and differentiation of many types of cells, including stem cells. High plasticity, low immunogenicity and immunomodulation ability of mesenchymal stem cells derived from human endometrium (eMSCs) allow them to be considered a prominent tool for regenerative medicine. Here, we examined the role of ATP in the proliferation and migration of human eMSCs. Using a wound healing assay, we showed that ATP‐induced activation of purinergic receptors suppressed the migration ability of eMSCs. We found the expression of one of the ATP receptors, the P2X₇ receptor in eMSCs. In spite of this, cell activation with specific P2X₇ receptor agonist, BzATP did not significantly affect the cell migration. The allosteric P2X₇ receptor inhibitor, AZ10606120 also did not prevent ATP‐induced inhibition of cell migration, confirming that inhibition occurs without P2X₇ receptor involvement. Flow cytometry analysis showed that high concentrations of ATP did not have a cytotoxic effect on eMSCs. At the same time, ATP induced the cell cycle arrest, suppressed the proliferative and migration capacity of eMSCs and therefore could affect the regenerative potential of these cells.     

2′, 3′-<Emphasis Type="Italic">O</Emphasis>-(4-benzoylbenzoyl)–ATP-mediated calcium signaling in rat glioma C6 cells: role of the P2Y<Subscript>2</Subscript> nucleotide receptor

In this study, we examined the response of glioma C6 cells to 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) and showed that the BzATP-induced calcium signaling does not involve the P2X₇ receptor activity. We show here that in the absence of extracellular Ca²⁺, BzATP-generated increase in [Ca²⁺]_i via Ca²⁺ release from intracellular stores. In the presence of calcium ions, BzATP established a biphasic Ca²⁺ response, in a manner typical for P2Y receptors. Brilliant Blue G, a selective antagonist of the rat P2X₇ receptor, did not reduce any of the two components of the Ca²⁺ response elicited by BzATP. Periodate-oxidized ATP blocked not only BzATP- but also UTP-induced Ca²⁺ elevation. Moreover, BzATP did not open large transmembrane pores. What is more, a cross-desensitization between UTP and BzATP occurred, which clearly shows that in glioma C6 cells BzATP activates most likely the P2Y₂ but not the P2X₇ receptors.     

氨磷汀对氧糖剥夺引起的PC12细胞缺血再灌注损伤的保护作用研究

目的：研究氨磷汀对体外培养的神经元样细胞的缺血再灌注损伤的保护作用,为其最终用于临床脑缺血的治疗打下基础。方法：体外培养的PC12细胞氧糖剥夺4h后复氧复糖,给予不同浓度的氨磷汀处理,20h后镜下观察细胞形态学变化,用MTT和LDH检测细胞活力和损伤情况,免疫荧光染色观察凋亡细胞,流式细胞仪计数凋亡细胞的比例。结果：高浓度氨磷汀对正常PC12细胞活力有抑制作用（P〈0.05）,而低浓度则无。氨磷汀可以提高缺血再灌注损伤PC12细胞活力（P〈0.05）,减少LDH释放（P〈0.05）,保护细胞正常形态,抑制细胞凋亡（P〈0.05）。结论：氨磷汀对氧糖剥夺引起的神经元样细胞的缺血再灌注损伤具有保护作用。     

ATP receptors in pain sensation: Involvement of spinal microglia and P2X<Subscript>4</Subscript> receptors

There is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. At first, it was thought that ATP was simply involved in acute pain, since ATP is released from damaged cells and excites directly primary sensory neurons by activating their receptors. However, neither blocking P2X/Y receptors pharmacologically nor suppressing the expression of P2X/Y receptors molecularly in sensory neurons or in the spinal cord had an effect on acute physiological pain. The focus of attention now is on the possibility that endogenous ATP and its receptor system might be activated in pathological pain states, particularly in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. An important advance in our understanding of the mechanisms involved in neuropathic pain has been made by a recent work demonstrating the crucial role of ATP receptors (i.e., P2X₃ and P2X₄ receptors). In this review, we summarize the role of ATP receptors, particularly the P2X₄ receptor, in neuropathic pain. The expression of P2X₄ receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X₄ receptors produce a reduction of the neuropathic pain behaviour. Understanding the key roles of ATP receptors including P2X₄ receptors may lead to new strategies for the management of neuropathic pain.     

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。     

miR-146a down-regulation alleviates H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced cytotoxicity of PC12 cells by regulating MCL1/JAK/STAT pathway

Oxidative stress and miRNAs have been confirmed to play an important role in neurological diseases. The study aimed to explore the underlying effect and mechanisms of miR-146a in H₂O₂-induced injury of PC12 cells. Here, PC12 cells were stimulated with 200 μM of H₂O₂ to construct oxidative injury model. Cell injury was evaluated on the basis of the changes in cell viability, migration, invasion, apoptosis, and DNA damage. Results revealed that miR-146a expression was up-regulated in H₂O₂-induced PC12 cells. Functional analysis showed that down-regulation of miR-146a alleviated H₂O₂-induced cytotoxicity in PC12 cells. Dual-luciferase reporter and western blot assay verified that MCL1 was a direct target gene of miR-146a. Moreover, anti-miR-146a-mediated suppression on cell cytotoxicity was abated following MCL1 knockdown in H₂O₂-induced PC12 cells. Furthermore, MCL1 activated JAK/STAT signaling pathway and MCL1 overexpression attenuated H₂O₂-induced cytotoxicity in PC12 cells by JAK/STAT signaling pathway. In conclusion, this study suggested that suppression of miR-146a abated H₂O₂-induced cytotoxicity in PC12 cells via regulating MCL1/JAK/STAT pathway.     

Transgenic neuronal overexpression reveals that stringently regulated p23 expression is critical for coordinated movement in mice

Background

Previous studies indicate a role of P2X₇ receptors in processes that lead to neuronal death. The main objective of our study was to examine whether genetic deletion or pharmacological blockade of P2X₇ receptors influenced dopaminergic cell death in various models of Parkinson's disease (PD).

Results

mRNA encoding P2X₇ and P2X₄ receptors was up-regulated after treatment of PC12 cells with 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). P2X₇ antagonists protected against MPTP and rotenone induced toxicity in the LDH assay, but failed to protect after rotenone treatment in the MTT assay in PC12 cells and in primary midbrain culture. In vivo MPTP and in vitro rotenone pretreatments increased the mRNA expression of P2X₇ receptors in the striatum and substantia nigra of wild-type mice. Basal mRNA expression of P2X₄ receptors was higher in P2X₇ knockout mice and was further up-regulated by MPTP treatment. Genetic deletion or pharmacological inhibition of P2X₇ receptors did not change survival rate or depletion of striatal endogenous dopamine (DA) content after in vivo MPTP or in vitro rotenone treatment. However, depletion of norepinephrine was significant after MPTP treatment only in P2X₇ knockout mice. The basal ATP content was higher in the substantia nigra of wild-type mice, but the ADP level was lower. Rotenone treatment elicited a similar reduction in ATP content in the substantia nigra of both genotypes, whereas reduction of ATP was more pronounced after rotenone treatment in striatal slices of P2X₇ deficient mice. Although the endogenous amino acid content remained unchanged, the level of the endocannabinoid, 2-AG, was elevated by rotenone in the striatum of wild-type mice, an effect that was absent in mice deficient in P2X₇ receptors.

Conclusions

We conclude that P2X₇ receptor deficiency or inhibition does not support the survival of dopaminergic neurons in an in vivo or in vitro models of PD.