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OBJECTIVE@#To investigate the effect of interference of P2X4 receptor expression in tumor-associated macrophages (TAMs) on invasion and migration of glioma cells.@*METHODS@#C57BL/6 mouse models bearing gliomas in the caudate nucleus were examined for glioma pathology with HE staining and expressions of Iba-1 and P2X4 receptor with immunofluorescence assay. RAW264.7 cells were induced into TAMs using conditioned medium from GL261 cells, and the changes in mRNA expressions of macrophage polarization-related markers and the mRNA and protein expressions of P2X4 receptor were detected with RT-qPCR and Western blotting. The effect of siRNA-mediated P2X4 interference on IL-1β and IL-18 mRNA and protein expressions in the TAMs was detected with RT-qPCR and Western blotting. GL261 cells were cultured in the conditioned medium from the transfected TAMs, and the invasion and migration abilities of the cells were assessed with Transwell invasion and migration experiment.@*RESULTS@#The glioma tissues from the tumor-bearing mice showed a significantly greater number of Iba-1-positive cells, where an obviously increased P2X4 receptor expression was detected (P=0.001), than the brain tissues of the control mice (P < 0.001). The M2 macrophage markers (Arg-1 and IL-10) and M1 macrophage markers (iNOS and TNF-α) were both significantly up-regulated in the TAMs derived from RAW264.7 cells (all P < 0.01), but the up-regulation of the M2 macrophage markers was more prominent; the expression levels of P2X4 receptor protein and mRNA were both increased in the TAMs (P < 0.05). Interference of P2X4 receptor expression significantly lowered the mRNA(P < 0.01)and protein (P < 0.01, P < 0.05)expression levels of IL-1β and IL-18 in the TAMs and obviously inhibited the ability of the TAMs to promote invasion and migration of the glioma cells (P < 0.05).@*CONCLUSION@#Interference of P2X4 receptor in the TAMs suppresses the migration and invasion of glioma cells possibly by lowering the expressions of IL-1β and IL-18.
Subject(s)
Animals , Mice , Culture Media, Conditioned , Glioma , Interleukin-18 , Mice, Inbred C57BL , RNA, Messenger , Receptors, Purinergic P2X4/metabolism , Tumor-Associated MacrophagesABSTRACT
Neuropathic pain is a typical chronic pain that has a great impact on the psychology and physiology of patients. Recent studies have shown that spinal microglial activation and the up-regulated expression of P2X4 receptor on its surface were occured after peripheral nerve injury. P2X4 receptor is an ionic subtype of ATP receptor, and was regulated by various factors under pathological conditions of nerve injury, such as chemokines, extracellular matrix fibronectin and interferon regulatory factor 5. Inhibition of microglial activation and P2X4 receptor expression can reduce neuropathic pain. Therefore, this article mainly describes the research progress of P2X4 receptor in the pathogenesis of neuropathic pain.
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Objective To investigate the effect of hyperbaric oxygen(HBO)on p38 MAPK signal pathway in neuropathic pain,and explore its mechanism. Methods The experiment was divided into two parts with thirty SD rats in each part. Each part was then randomly divided into 3 groups:sham operation group(S group),chronic constriction group(CCI group)and hyperbaric oxygen group(HBO group),with 10 rats in each group. Part one:Pain?related behavior were detected on 3 day,7 day,14 day,28 day after operation. Phosphorylative p38 was detected by Western blot method and the P2X4 receptor was detected by immunohistochemistry method on the 28th day. Part two:All the rats were treated with SB203580. Pain?related behavior were detected on 3 day,7 day,14 day,28 day after operation. P2X4 receptor was detected by immunohistochem?istry method on the 28th day. Results CCI and HBO groups were significantly lower than S group in pain?related behaviors(P0.05). Though the expression of P2X4 receptor in CCI group and HBO group was significantly higher than that in S group(P0.05). Conclusion Hyperbaric oxygen therapy can affect the expression of p38 MAPK through P2X4 receptor in rats of neuropathic pain.
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AIM:To investigate the effects of P2X4 receptor on peri-sciatic administration of recombinant rat TNF-α(rrTNF)-induced mechanical allodynia.METHODS:Male Sprague-Dawley rats (180~200 g) were used in the experiments.The levels of P2X4 receptor on day 3, day 7 and day 14 after peri-sciatic administration of rrTNF were exam-ined by Western blot, and the location of P2X4 receptor in the spinal dorsal horn was observed by double immunofluores-cence staining.The changes of 50%paw-withdrawal thresholds of the rat were detected by behavioral test, and the level of TNF-αin the spinal dorsal horn was also examined by Western blot when TNP-ATP was intrathecally injected before the ad-ministration of rrTNF.RESULTS:Compared with control group, the expression of P2X4 receptor in the spinal dorsal horn on the ipsilateral side significantly increased on day 3, day 7 and day 14 (P<0.01) after rrTNF (100 ng/L) administra-tion.P2X4 receptor was co-localized only with microglia, but not with neurons or astrocytes.Intrathecal injection of TNP-ATP before rrTNF administration prevented mechanical allodynia induced by rrTNF and inhibited the upregulation of TNF-αin the spinal dorsal horn.CONCLUSION:P2X4 receptors in microglia may be involved in rrTNF-induced mechanical allodynia by the upregulation of TNF-αin the spinal dorsal horn.
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O receptor P2X4 (canal iônico controlado por adenosina-5'-trifosfato-ATP) está amplamente distribuído no sistema nervoso central e, após sua ativação, pode regular os níveis de cálcio intracelulares via permeação direta e por ativação de canais de cálcio voltagem-dependentes. Tem sido proposto que a atividade do receptor pode ser importante na plasticidade sináptica. Tendo em vista a importância do receptor P2X4, sobretudo na fisiologia do sistema nervoso central, é útil caracterizá-lo farmacologicamente e entender os mecanismos moleculares que regulam sua atividade. Examinamos o papel que resíduos específicos N- e C-terminais desempenham na atividade do receptor P2X4 humano, combinando técnicas de biologia molecular, bioquímica e patch-clamp em células de rim de embrião humano (células HEK-293T). Células HEK-293T expressando o receptor P2X4 wild-type apresentaram correntes iônicas, cujas amplitudes dependeram da concentração de ATP, fornecendo um valor de EC50 de 1,37 ± 0,21 µM. Os receptores mutantes E14A e D16A exibiram respostas ao ATP equiparáveis àquelas do receptor selvagem, ao passo que os mutantes Y15A e T17A não foram funcionais, apesar de serem expressos na membrana plasmática das células. A inibição de tirosina fosfatases por pervanadato diminuiu fortemente correntes induzidas por ATP. Subsequente análise de citometria de fluxo na presença de um anticorpo contra resíduos de fosfotirosina indicaram que, entre as células que expressam o receptor P2X4, a percentagem de células fosfo-tirosina-positivas é a mesma para os mutantes Y372A (86 ± 10%) e Y378A (79 ± 6.9%), mas substancialmente menor para os mutantes Y15A (35 ± 12%), Y367A (48 ± 6.4%) e Y372F (31 ± 1.7%), quando comparados com células que expressam o receptor wild-type (76 ± 5.6%). Resultados semelhantes foram obtidos quando quantificamos a expressão relativa de proteínas fosforiladas em resíduos de tirosina e expressamos através dos valores de intensidade de fluorescência média. Ensaios de western-blot revelaram que mesmo o mutante T17A é fosforilado em resíduos de treonina, sugerindo que o receptor P2X4 contém outros sítios de fosforilação. Entretanto, nenhum sinal de fosfotirosina foi detectado no receptor wild-type e nos mutantes, em que resíduos de tirosina foram substituídos por alanina ou fenilalanina. Não parece ser o resíduo Y15 o alvo de tal fosforilação, cabendo a ele um papel estrutural mais importante. Nossos dados também sugerem que a fosforilação em resíduos de tirosina de proteínas intermediárias regula a atividade do receptor P2X4
The human P2X4 receptor (ATP-gated ion channel) is widely distributed in the CNS and, after activation, participates in regulation of levels of intracellular calcium through direct permeation and activation of voltage-dependent calcium channels with well-defined functions including synaptic plasticity. Given the importance of the P2X4 receptor, especially in CNS physiology, we investigated the role that specific N- and C-termini residues play in human P2X4 receptor activity, by combining techniques of molecular biology, biochemistry and patch-clamping in human embryonic kidney cells (HEK-293T cells). HEK-293T cells expressing the wild-type P2X4 receptor showed ionic currents whose amplitudes depended on the ATP concentration, providing an EC50 value of 1.37 ± 0.21 mM. E14A and D16A receptor mutants exhibited responses to ATP comparable to those ones of wild-type receptor, whereas Y15A and T17A mutants were not functional, despite being expressed in the plasma membrane of cells. The inhibition of tyrosine phosphatases by pervanadate decreased strongly ATP-induced currents. Subsequent flow cytometry analysis in the presence of an antibody against phosphotyrosine residues indicated that, among the cells that express the P2X4 receptor, the percentage of phosphotyrosine-positive cells was the same for Y372A (86 ± 10%) and Y378A (79 ± 6.9%) mutants, however, substantially lower for Y15A (35 ± 12%), Y367A (48 ± 6.4%) and Y372F (31 ± 1.7%) mutants when compared with cells expressing the wild-type receptor (76 ± 5.6%). Similar results were obtained by quantifying the relative expression of phosphotyrosine proteins. Western blot assays revealed that even the T17A mutant was phosphorylated at threonine residues, suggesting that the human P2X4 receptor also contains further phosphorylation sites. However, no phosphotyrosine-antibody signal was detected in the wild-type receptor and mutants in which tyrosine residues were replaced by alanine or phenylalanine. The residue Y15 is supposedly not the target of such phosphorylation, despite its important structural role. However, the present work indicates that tyrosine phosphorylation of intermediate signaling proteins regulates P2X4 receptor activity
Subject(s)
Receptors, Purinergic P2X4/genetics , Threonine/analysis , Tyrosine/analysis , Blotting, Western/instrumentation , Central Nervous System/physiology , Flow Cytometry/methods , Patch-Clamp Techniques/methods , PhosphorylationABSTRACT
Objective: To observe the effect intraperitoneal injection of low dose ketamine on thermal hyperalgesia and expression of P2X4 receptor in spinal dorsal horn of rats with chronic constriction injury (CCI) of sciatic nerve, and to explore the potential role of P2X4, receptor in the neuropathic pain. Methods: Totally 24 Sprague-Dawley rats were randomly divided into 3 groups (n=8): group S (sham group), group C: CCI + normal saline; and group K:CCI+ketamine (10mg · kg-1). Rat CCI model was used in the latter 2 groups. Three days after operation the thermal withdrawal latency (TWL) was determined to confirm the thermal hyperalgesia. Rats in group K were given low dose of ketamine (10mg · kg-1) and those in group C were given the same volume of normal saline for 7 days after operation. Animals in group S only had sciatic nerve exposed, with no ligation or drugs. TWL was determined 1 day before and 1, 3, 7 days after the operation. The expression of P2X4 receptor was assessed 7 days after the operation using immunohistochemistry. Results: The TWL values were similar between the 3 groups before operation. The value in group S was slightly decreased after operation compared with before operation. Compared with the pre-operation, group S, and group C, the TWL value of group K began to gradually increase 3 days after operation till day 7 after operation (P<0.05). On day 7 after operation, the TWL value was significantly higher than group C (P<0.05), but was still lower than that in group S (P<0.05). Immunohistochemistry showed that the expression of P2X4 receptor in group C, K were significantly higher than that of group S (P<0.01) and the expression in group K was significantly lower than that in group C (P<0.05). Conclusion: Intraperitoneal injection of ketamine can partly relieve the thermal hyperalgesia in rats with CCI of sciatic nerve, which might be related to the inhibition of P2X4 receptor expression in the spinal dorsal horn.