Interference of P2X4 receptor expression in tumor-associated macrophages suppresses migration and invasion of glioma cells / 南方医科大学学报

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.

Overexpression of Purinergic P2X4 Receptors in Hippocampus Rescues Memory Impairment in Rats with Type 2 Diabetes / 神经科学通报·英文版

Purinergic receptors have been reported to be involved in brain disorders. In this study, we explored their roles and mechanisms underlying the memory impairment in rats with type 2 diabetes mellitus (T2DM). T2DM rats exhibited a worse performance in the T-maze and Morris water maze (MWM) than controls. Microglia positive for P2X purinoceptor 4 (P2X4R) in the hippocampus were reduced and activated microglia were increased in T2DM rats. Long Amplicon PCR (LA-PCR) showed that DNA amplification of the p2x4r gene in the hippocampus was lower in T2DM rats. Minocycline significantly reduced the number of activated microglia and the mean distance traveled by T2DM rats in the MWM. Most importantly, P2X4R overexpression suppressed the activated microglia and rescued the memory impairment of T2DM rats. Overall, T2DM led to excessive activation of microglia in the hippocampus, partly through the DNA damage-mediated downregulation of P2X4Rs, thus contributing to memory impairment.

Electroacupuncture at "Jiaji" (EX-B2) induced down-regulation of OX-42 and P2X4 in lumbar spinal cord contributes to its analgesic after-effect in rats with chronic constriction injury / 针刺研究

OBJECTIVE: To investigate the effect of electroacupuncture (EA) at "Jiaji" (EX-B2) at different time points on the expression of OX-42 (a monoclonal antibody with specific expression of complement receptor-3 in spinal microglial cells) and purinergic receptor P2X4 (P2X4) in rats with chronic constriction injury (CCI) of the sciatic nerve, as well as the possible after-effect mechanism of EA analgesia in neuropathic pain. METHODS: Sprague-Dawley rats were randomly divided into blank group, model group, immediately after EA group, 0.5-hour after EA group, 1-hour after EA group, 2-hour after EA group, 4-hour after EA group, 12-hour after EA group, and 24-hour after EA group, with 6 rats in each group. The rats in the model group and the EA groups were used to establish a model of CCI-induced neuropathic pain, and those in the immediately after EA group and the 0.5, 1, 2, 4, 12 and 24 hours after EA groups were treated with EA at bilateral L3 and L5 "Jiaji" points for 20 min after 7 d of modeling. Samples were collected immediately and at 0.5, 1, 2, 4, 12, and 24 hours after EA, and for the rats in the blank group and the model group, samples were collected after fixation the rats for 20 min. Heat pain threshold was observed before and after intervention, and immunohistochemistry was used to measure the protein expression of OX-42 and P2X4 in the spinal cord lumbar enlargement. RESULTS: After 7 days of modeling (before intervention), compared with the blank group, the heat pain threshold had a significant reduction in the model group and the EA groups (P<0.01). Compared with the model group after intervention, the immediately after EA group and the 0.5, 1 and 2 hours after EA groups had a significant increase in heat pain threshold (P<0.05). Compared with the model group, immediately after EA, the 0.5, 1 and 2 hours after EA groups had a significant reduction in the protein expression of OX-42 (P<0.01), and immediately after EA, the 0.5 and 1 hour after EA groups had a significant reduction in the protein expression of P2X4 (P<0.01). CONCLUSION: EA at "Jiaji" points can significantly increase heat pain threshold and down-regulate the protein expression of OX-42 and P2X4 in the spinal cord of CCI rats. The analgesic effect can last for 2 h.

Dexmedetomidine attenuates P2X4 and NLRP3 expression in the spine of rats with diabetic neuropathic pain

Abstract Purpose: To evaluate the effects of Dexmedetomidine (Dex) on spinal pathology and inflammatory factor in a rat model of Diabetic neuropathic pain (DNP). Methods: The rats were divided into 3 groups (eight in each group): normal group (N group), diabetic neuropathic pain model group (DNP group), and DNP model with dexmedetomidine (Dex group). The rat model of diabetes was established with intraperitoneal streptozotocin (STZ) injections. Nerve cell ultrastructure was evaluated with transmission electron microscopy (TEM). The mechanical withdrawal threshold (MWT) and motor nerve conduction velocity (MNCV) tests documented that DNP rat model was characterized by a decreased pain threshold and nerve conduction velocity. Results: Dex restored the phenotype of neurocytes, reduced the extent of demyelination and improved MWT and MNCV of DNP-treated rats (P=0.01, P=0.038, respectively). The expression of three pain-and inflammation-associated factors (P2X4, NLRP3, and IL-IP) was significantly upregulated at the protein level in DNP rats, and this change was reversed by Dex administration (P=0.0022, P=0.0092, P=0.0028, respectively). Conclusion: The P2X4/NLRP3 signaling pathway is implicated in the development and presence of DNP in vivo, and Dex protects from this disorder.

The effect of overxpression of P2X4 on cell apoptosis in a rat model of Parkinson Disease / 中国神经精神疾病杂志

Objective To investigate the effect of overexpression of P2X4 gene on cell apoptosis in 6-OHDA (6-hydroxydopamine, 6-OHDA) induced PD rat model. Methods One hundred twenty male Wistar rats were randomly divided into 6 groups: 6-OHDA group, control group, objective RNA-P2X4 + 6-OHDA group, target gene RNA-P2X4 group, P2X4-NC + 6-OHDA group and negative virus P2X4-NC only group. Lentivirus or negative virus carrying the target gene and or 6-OHDA or the same amount of physiological saline were injected into the left substantia nigra of rats according to the groups. The number of dopaminergic neurons in substantia nigra was measured by immunofluorescence method.The expression levels of P2X4R,caspase-1 and NLRP3 in substantia nigra were detected by Western blot assay. Results Compared with P2X4-NC + 6-OHDA group, the expression of protein P2X4R (1.099 ± 0.05569 vs. 0.7821 ± 0.02008, P=0.0003 ), NLRP3 (0.9875 ± 0.01932 vs. 0.6645 ± 0.01747. P<0.0001), caspase-1 (0.9948 ± 0.01788 vs. 0.8276 ± 0.04543, P<0.0001) increased significantly in RNA-P2X4+6-OHDA group. Conclusion overexpression of P2X4 gene can increase the expression of NLPR3 and caspase-1 protein and promote apoptosis in PD rats.

Research progress of microglia-P2X4 receptor in neuropathic pain / 医学研究生学报

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.

Effects of lappaconitine on pain and inflammatory response of severely burned rats and the mechanism / 中华烧伤杂志

Objective@#To explore the effects of lappaconitine (LA) on pain and inflammatory response of severely burned rats and the mechanism.@*Methods@#Forty SD rats were divided into healthy+ normal saline group, sham injury+ normal saline group, pure burn group, burn+ LA group, and healthy+ LA group according to the random number table (the same dividing method below), with 8 rats in each group. Rats in pure burn and burn+ LA groups were inflicted with about 32% total body surface area deep partial-thickness scald (hereinafter referred to as burn) on the back and right hind. Rats in sham injury+ normal saline group were sham injured. Rats in burn+ LA group were intraperitoneally injected with 1 g/L LA solution in the dosage of 4 mL/kg at 2.0 h before injury and post injury hour (PIH) 0 (immediately), 24.0, 48.0, and 72.0. Rats in healthy+ LA group were intraperitoneally injected with LA solution in the same dose at the same time points as above, and rats in healthy+ normal saline and sham injury+ normal saline groups were intraperitoneally injected with normal saline in the dose of 4 mL/kg at the same time points as above. At 1.5 h before injury and PIH 12.5, 24.5, 36.5, 48.5, and 72.5, the paw withdrawal mechanical threshold (PWMT) of injured rats was detected, and their pain behaviors were observed. The same observation and detection were conducted in rats without injury in the two groups at the same time points as above. Another 32 SD rats were divided into normal saline group, trinitrophenyl (TNP)-ATP group, minocyline group, pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS) group, with 8 rats in each group, and all the rats were inflicted with the same burn injury as above. At PIH 48.0, rats in normal saline group were intrathecally injected with 10 μL normal saline; rats in TNP-ATP group were intrathecally injected with 10 μL TNP-ATP in the concentration of 30 nmol/μL; rats in minocyline group were intrathecally injected with 10 μL minocyline in the concentration of 5 g/L; rats in PPADS group were intrathecally injected with 10 μL PPADS in the concentration of 10 nmol/μL. The PWMT of rats was detected at 0.5 h before injection and 0.5 h after. At PIH 72.5, the tissue in the dorsal horn of spinal cord of rats in sham injury+ normal saline, pure burn, and burn+ LA groups was harvested to observe the co-expression of P2X4 receptor and OX42 receptor with immunofluorescent staining and to observe the expression of P2X4 receptor and count the positive cells with immunohistochemical staining. The venous blood was harvested for determination of serum content of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) with enzyme-linked immunosorbent assay. The same observation and determination were conducted in rats without injury in the two groups at the same time point as above. Data were processed with one-way analysis of variance, analysis of variance for repeated measurement, SNK test, paired t test, and Bonferroni correction.@*Results@#(1) There were no abnormal activity in rats of healthy+ normal saline, sham injury+ normal saline, healthy+ LA groups at all time points. Until PIH 72.5, rats in pure burn group were in poor mental state; red and swollen manifestation and blister were observed in burn wounds on the back and right hind; imbalance in gait, lick, bite, and scratch were observed occasionally. Fewer behaviors such as lick, bite, and limp were observed in rats in burn+ LA group than in pure burn group, and the red and swollen manifestation in wounds of rats in burn+ LA group dissipated faster than that in pure burn group. (2) At 1.5 h before injury, there were no significant differences in the PWMT values of rats in healthy+ normal saline, sham injury+ normal saline, pure burn, burn+ LA, and healthy+ LA groups (F=0.106, P>0.05). PWMT values of rats in pure burn group were significantly lower than those in the other 4 groups at all post injury time points (with P values below 0.05). PWMT values of rats in burn+ LA group were significantly lower than those in healthy+ normal saline, sham injury+ normal saline, and healthy+ LA groups at all post injury time points (with P values below 0.05). (3) At 0.5 h before injection, PWMT values of rats in normal saline, TNP-ATP, PPADS, and minocyline groups were close, respectively 15.3±0.8, 15.1±1.0, 15.3±0.9, and 15.6±1.1 (F=0.343, P>0.05). At 0.5 h after injection, PWMT values of rats in normal saline group and PPADS group were respectively 15.2±1.2 and 14.8±1.0, which were significantly lower than 20.8±1.4 and 26.3±1.0 in TNP-ATP group and minocyline group respectively (with P values below 0.05). PWMT values of rats in normal saline and PPADS groups were similar before and after injection (with t values respectively 0.073 and -0.772, P values above 0.05), while those of rats in TNP-ATP and minocyline groups were higher after injection than before injection (with t values respectively -10.180 and -20.813, P values below 0.01). (4) At PIH 72.5, co-expression of P2X4 receptor and OX42 receptor was observed in a few microglias of rats in healthy+ normal saline, sham injury+ normal saline, and healthy+ LA groups, while co-expression of P2X4 receptor and OX42 receptor was observed in a large number of microglias of rats in pure burn and burn+ LA groups. At PIH 72.5, more P2X4 receptor positive cells were observed in rats in pure burn group than in the other 4 groups (with P values below 0.05), and more P2X4 receptor positive cells were observed in rats in burn+ LA group than in healthy+ normal saline, sham injury+ normal saline, and healthy+ LA groups (with P values below 0.05). (5) At PIH 72.5, the serum content of TNF-α and IL-1β of rats in pure burn group was significantly higher than that in the other 4 groups (with P values below 0.001). The serum content of TNF-α and IL-1β of rats in burn+ LA group was significantly lower than that in healthy+ normal saline, sham injury+ normal saline, and healthy+ LA groups (with P values below 0.001).@*Conclusions@#LA has significant analgesic effects on severely burned rats, and it can ameliorate the excessive inflammational situation. The mechanism may be related to its inhibition of expression of P2X4 receptor in microglias in the dorsal horn of spinal cord and reduction in the release of inflammatory factors TNF-α and IL-1β.

Role of P2X4 receptor,a subtype of ionotropic purinoceptors, in central nervous system and its potential role as a drug target for treating neurological disorders / 中国药理学与毒理学杂志

Adenosine 5-triphosphate is not only the source of cellular energy,but also the extracel-lular signal molecule that binds two different classes of ATP receptors:ionotropic P2X receptors and G-protein-coupled P2Y receptors.P2X4 receptor is one of the ionotropic P2X receptors widely expressed in the central nervous system(CNS).This subtype of the ionotropic P2X receptor is implicated in many physiological and pathological functions in the CNS, including chronic pain, alcohol abuse, seizures, Alzheimer′s and Parkinson′s diseases,and stroke.Therefore,P2X4 receptor has attracted attention as a promising drug target.Here,we review the findings of research or the physiological and pathological functions of P2X4 in the CNS. Recent progress in the development of new pharmacological agents of P2X4,such as selective antagonists,is also presented here.

Effect of Hyperbaric Oxygen on p38 MAPK Signal Transduction Pathway in Neuropathic Rats / 中国医科大学学报

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.

Effects of P2X4 receptor in spinal microglia on rrTNF-induced pathologi-cal pain / 中国病理生理杂志

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.

Identificação de resíduos de treonina e tirosina importantes na regulação da atividade do receptor P2X4 humano através de mutagênese sítio-dirigida / Identification of threonine and tyrosine residues important for human P2X4 receptor activity by site-directed mutagenesis

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

Low-dose ketamine inhibits expression of P2X4 receptor in spinal dorsal horn of rats with chronic neuropathic pain / 第二军医大学学报

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.