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目的研究代谢性谷氨酸受体第5亚型（mGluR5）在牙髓各部位中的表达及分布。方法收集2009年7月至2010年1月山东大学口腔医院口腔颌面外科因正畸或其他治疗需要而拔除的健康前磨牙或第三磨牙5例,制成一系列石蜡切片,利用免疫组化方法检测牙髓各部位mGluR5的表达及分布情况,利用图像分析系统对其表达强度进行半定量分析,探讨mGluR5在牙髓中的作用和意义。结果正常牙髓从冠部、颈部到根部牙髓成牙本质细胞中mGluR5表达均呈阳性,且由冠部、颈部到根部mGluR5表达强度依次降低。结论mGluR5在牙髓疼痛传递过程中可能具有一定的作用。     

沉默mGluR5对卵巢癌SKOV3细胞生物学特性的影响及其机制研究

目的 探讨小干扰RNA（siRNA）靶向代谢型谷氨酸受体5（mGluR5）对卵巢癌SKOV3细胞增殖、凋亡和迁移能力的影响及可能的机制。方法 采用靶向mGluR5表达的特异siRNA 转染卵巢癌SKOV3细胞（转染组），同时设置转染无义序列的阴性对照组。转染48 h，采用实时荧光定量PCR（QPCR）检测siRNA对mGluR5的沉默效果；采用CCK-8法、EdU细胞荧光染色实验、Hoechst 33342细胞染色实验、划痕实验检测沉默mGluR5表达后SKOV3细胞的活性、增殖、凋亡和迁移情况；采用Western blotting检测PTEN／Akt信号通路相关蛋白的表达情况。结果 QPCR检测显示，转染组中mGluR5 mRNA的表达水平降至（18.3±2.3）％，显著低于阴性对照组（P＜0.05）。CCK-8法检测显示，转染72 h阴性对照组的吸光值为4.1±0.2，高于转染组的2.4±0.3（P＜0.05）。EdU免疫荧光染色实验显示，转染48 h阴性对照组的细胞增殖率为（22.4±2.3）％，高于转染组的（9.2±1.2）％（P＜0.05）。Hoechst 33342细胞染色实验显示，转染48 h阴性对照组的细胞凋亡率为（6.2±1.3）％，低于转染组的（28.2±2.2）％（P＜0.05）。划痕实验显示，阴性对照组的相对划痕愈合比例为（100.0±2.1）％，显著高于转染组的（48.6±4.3）％（P＜0.05）。Western blotting检测显示，转染组PTEN蛋白的表达增加，p-Akt蛋白的表达降低（P＜0.05）；而Akt蛋白无明显变化（P＞0.05）。结论 沉默mGluR5表达能显著抑制卵巢癌SKOV3细胞的增殖，促进凋亡，降低细胞迁移能力，该过程可能与激活PTEN／Akt信号通路有关。     

Blocking glutamate‐mediated signalling inhibits human melanoma growth and migration

Glutamate is an excitatory neurotransmitter that has been shown to regulate the proliferation, migration and survival of neuronal progenitors in the central nervous system through its action on metabotropic and ionotropic glutamate receptors (GluRs). Antagonists of ionotropic GluRs have been shown to cause a rapid and reversible change in melanocyte dendritic morphology, which is associated with the disorganization of actin and tubulin microfilaments in the cytoskeleton. Intracellular expression of microtubule‐associated protein (MAP) 2a affects the assembly, stabilization and bundling of microtubules in melanoma cells; stimulates the development of dendrites; and suppresses melanoma cell migration and invasion. In this study, we investigated the relationship between glutamate‐mediated signalling and microtubules, cell dendritic morphology and melanoma cell motility. We found that metabotropic GluR1 and N‐methyl‐d ‐aspartate receptor antagonists increased dendritic branching and inhibited the motility, migration and proliferation of melanoma cells. We also demonstrated that the invasion and motility of melanoma cells are significantly inhibited by the combination of increased expression of MAP2a and either metabotropic GluR1 or N‐methyl‐d ‐aspartate receptor antagonists. Moreover, the blockade of glutamate receptors inhibited melanoma growth in vivo. Collectively, these results demonstrate the importance of glutamate signalling in human melanoma and suggest that the blockade of glutamate receptors is a promising novel therapy for treating melanoma.     

Melatonin impedes Tet1‐dependent mGluR5 promoter demethylation to relieve pain

Melatonin (N‐acetyl‐5‐methoxytryptamine)/MT2 receptor‐dependent epigenetic modification represents a novel pathway in the treatment of neuropathic pain. Because spinal ten‐eleven translocation methylcytosine dioxygenase 1 (Tet1)‐dependent epigenetic demethylation has recently been linked to pain hypersensitivity, we hypothesized that melatonin/MT2‐dependent analgesia involves spinal Tet1‐dependent demethylation. Here, we showed that spinal Tet1 gene transfer by intrathecal delivery of Tet1‐encoding vectors to naïve rats produced profound and long‐lasting nociceptive hypersensitivity. In addition, enhanced Tet1 expression, Tet1‐metabotropic glutamate receptor subtype 5 (mGluR5) promoter coupling, demethylation at the mGluR5 promoter, and mGluR5 expression in dorsal horn neurons were observed. Rats subjected to spinal nerve ligation and intraplantar complete Freund's adjuvant injection displayed tactile allodynia and behavioral hyperalgesia associated with similar changes in the dorsal horn. Notably, intrathecal melatonin injection reversed the protein expression, protein‐promoter coupling, promoter demethylation, and pain hypersensitivity induced by Tet1 gene transfer, spinal nerve ligation, and intraplantar complete Freund's adjuvant injection. All the effects caused by melatonin were blocked by pretreatment with a MT2 receptor‐selective antagonist. In conclusion, melatonin relieves pain by impeding Tet1‐dependent demethylation of mGluR5 in dorsal horn neurons through the MT2 receptor. Our findings link melatonin/MT2 signaling to Tet1‐dependent epigenetic demethylation of nociceptive genes for the first time and suggest melatonin as a promising therapy for the treatment of pain.     

Positive AMPA receptor modulation in the treatment of neuropsychiatric disorders: A long and winding road

Glutamatergic transmission is widely implicated in neuropsychiatric disorders, and the discovery that ketamine elicits rapid-acting antidepressant effects by modulating α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) signaling has spurred a resurgence of interest in the field. This review explores agents in various stages of development for neuropsychiatric disorders that positively modulate AMPARs, both directly and indirectly. Despite promising preclinical research, few direct and indirect AMPAR positive modulators have progressed past early clinical development. Challenges such as low potency have created barriers to effective implementation. Nevertheless, the functional complexity of AMPARs sets them apart from other drug targets and allows for specificity in drug discovery. Additional effective treatments for neuropsychiatric disorders that work through positive AMPAR modulation may eventually be developed.     

MTEP impedes the neuronal polarization and the activity of the Akt–NF‐κB pathway in rat hippocampal neurons

Metabotropic glutamate receptor 5 (mGluR5) is extensively involved in neural survival, differentiation, dendritic morphology, synaptic plasticity, and neural circuit formation. However, little is known about its role in neuronal polarization and axon outgrowth. In this study, we applied the selective agonist (RS)‐2‐chloro‐5‐hydroxyphenylglycine sodium salt and antagonist 3‐[(2‐methyl‐4‐thaizolyl) ethynyl] pyridine (MTEP) of mGluR5 to the cultured hippocampal neurons to observe the neuronal polarization and axon outgrowth, and further explored the possible intracellular signal transduction pathway. The results demonstrated that MTEP administration significantly attenuates the proportion of polarized neurons and the length of the axon, indicated by SMI312 (an axon marker) and Tuj‐1 (a marker of all the neurites) double‐labeling immunofluorescence. Western blot analysis showed that MTEP administration also inhibited the activation of AKT and nuclear translocation of nuclear factor‐κB (NF‐κB) p65, and decreased the phosphorylation of p65 as well. Furthermore, Akt inhibitor LY294002 treatment resulted in neuronal polarization delay and axon outgrowth retardation, while suppressing the phosphorylation and nuclear translocation of p65. We concluded that mGluR5 could regulate neuronal polarity and axon outgrowth during the morphological differentiation of rat developing neurons, and the intracellular signaling pathway of Akt–NF‐κB might be involved in the action of mGluR5. © 2016 Wiley Periodicals, Inc.     

Cone synapses in mammalian retinal rod bipolar cells

Some mammalian rod bipolar cells (RBCs) can receive excitatory chemical synaptic inputs from both rods and cones (DBC_R2), but anatomical evidence for mammalian cone‐RBC contacts has been sparse. We examined anatomical cone‐RBC contacts using neurobiotin (NB) to visualize individual mouse cones and standard immuno‐markers to identify RBCs, cone pedicles and synapses in mouse and baboon retinas. Peanut agglutinin (PNA) stained the basal membrane of all cone pedicles, and mouse cones were positive for red/green (R/G)‐opsin, whereas baboon cones were positive for calbindin D‐28k. All synapses in the outer plexiform layer were labeled for synaptic vesicle protein 2 (SV2) and PSD (postsynaptic density)‐95, and those that coincided with PNA resided closest to bipolar cell somas. Cone‐RBC synaptic contacts were identified by: (a) RBC dendrites deeply invaginating into the center of cone pedicles (invaginating synapses), (b) RBC dendritic spines intruding into the surface of cone pedicles (superficial synapses), and (c) PKCα immunoreactivity coinciding with synaptic marker SV2, PSD‐95, mGluR6, G protein beta 5 or PNA at cone pedicles. One RBC could form 0‐1 invaginating and 1‐3 superficial contacts with cones. 20.7% and 38.9% of mouse RBCs contacted cones in the peripheral and central retina (p < .05, n = 14 samples), respectively, while 34.4% (peripheral) and 48.5% (central) of cones contacted RBCs (p > .05). In baboon retinas (n = 4 samples), cone‐RBC contacts involved 12.2% of RBCs (n = 416 cells) and 22.5% of cones (n = 225 cells). This suggests that rod and cone signals in the ON pathway are integrated in some RBCs before reaching AII amacrine cells.     

Ammonia metabolism,the brain and fatigue; revisiting the link

This review addresses the ammonia fatigue theory in light of new evidence from exercise and disease studies and aims to provide a view of the role of ammonia during exercise. Hyperammonemia is a condition common to pathological liver disorders and intense or exhausting exercise. In pathology, hyperammonemia is linked to impairment of normal brain function and the onset of the neurological condition, hepatic encephalopathy. Elevated blood ammonia concentrations arise due to a diminished capacity for removal via the liver and lead to increased exposure of organs, such as the brain, to the toxic effects of ammonia. High levels of brain ammonia can lead to deleterious alterations in astrocyte morphology, cerebral energy metabolism and neurotransmission, which may in turn impact on the functioning of important signalling pathways within the neuron. Such changes are believed to contribute to the disturbances in neuropsychological function, in particular the learning, memory, and motor control deficits observed in animal models of liver disease and also patients with cirrhosis. Hyperammonemia in exercise occurs as a result of an increased production by contracting muscle, through adenosine monophosphate (AMP) deamination (the purine nucleotide cycle) and branched chain amino acid (BCAA) deamination prior to oxidation. Plasma concentrations of ammonia during exercise often achieve or exceed those measured in liver disease patients, resulting in increased cerebral uptake. In this article we propose that exercise-induced hyperammonemia may lead to concomitant disturbances in brain function, potentially through similar mechanisms underpinning pathology, which may impact on performance as fatigue or reduced function, especially during extreme exercise.     

Neurologic Autoimmunity in the Era of Checkpoint Inhibitor Cancer Immunotherapy

Neurologic autoimmune disorders in the context of systemic cancer reflect antitumor immune responses against onconeural proteins that are autoantigens in the nervous system. These responses observe basic principles of cancer immunity and are highly pertinent to oncological practice since the introduction of immune checkpoint inhibitor cancer therapy. The patient’s autoantibody profile is consistent with the antigenic composition of the underlying malignancy. A major determinant of the pathogenic outcome is the anatomic and subcellular location of the autoantigen. IgGs targeting plasma membrane proteins (eg, muscle acetylcholine receptor -IgG in patients with paraneoplastic myasthenia gravis) have pathogenic potential. However, IgGs specific for intracellular antigens (eg, antineuronal nuclear antibody 1 [anti-Hu] associated with sensory neuronopathy and small cell lung cancer) are surrogate markers for CD8⁺ T lymphocytes targeting peptides derived from nuclear or cytoplasmic proteins. In an inflammatory milieu, those peptides translocate to neural plasma membranes as major histocompatibility complex class I protein complexes. Paraneoplastic neurologic autoimmunity can affect any level of the neuraxis and may be mistaken for cancer progression. Importantly, these disorders generally respond favorably to early-initiated immunotherapy and cancer treatment. Small cell lung cancer and thymoma are commonly associated with neurologic autoimmunity, but in the context of checkpoint inhibitor therapy, other malignancy associations are increasingly recognized.