Uridine adenosine tetraphosphate acts as a pro-angiogenic factor in vitrothrough purinergic P2Y receptors

OBJECTIVE Uridine adenosine tetraphosphate(Up4A),a dinucleotide,contains both purine and pyrimidine moieties,and exerts its vascular influence via activation of purinergic receptors.Here,we aimed to investigate the effects of Up4 A on angiogenesis and the putative purinergic receptors(PR)involved in this process.METHODS Tubule formation assay was performed in 3D matrix system.In this assay,human umbilical vein endothelial cells(HUVECs)were co-cultured with pericytes with various Up4 A doses(0,1,2.5,5,10 and 20μmol·L-1)in the absence and presence of P2Y6 R antagonist MRS2578(10μmol·L-1)for 5d.Expression profile of PR subtypes and angiogenic factors was assessed in HUVECs by q-PCR with and without P2Y6 R antagonist.RESULTS No difference in initial tubule formation was detected between Up4 A stimulation and control conditions at day 2.In contrast,a significant increase in vascular density in response to Up4 A was observed at day 5.Up4 A at a dose of 2.5and 5μmol·L-1 promoted total tubule length(by-1.89 fold and-2.23fold),number of tubules(by-1.71 fold and-1.89fold)as well as number of junctions(by-2.24 fold and-2.80fold),all of which were inhibited by MRS2578.Further increase in Up4 A dose to10 and 20μmol·L-1 did not induce an increase in these vascular parameters as compared to non-treated controls.Moreover,Up4 A increased mRNA level of P2YRs(P2Y2R,P2Y4 R and P2Y6R)but not P2XR(P2X4R and P2X7R)or P1R(A2AR and A2BR),while Up4 A upregulated VEGFA and ANGPT1 but not VEGFR2,ANGPT2,Tie1 and Tie2at mRNA level.Transcriptional upregulation of P2 YRs and angiogenic factors by Up4 A was inhibited by MRS2578.CONCLUSION Up4 A is functionally capable of promoting tubule formation in vitro co-culture system.This process is likely mediated by activation of pyrimidine-favored P2 YRs but not P2 XR or P1 Rs,and involves stimulation of well known angiogenic factors.

Nuclear receptors and pathogenesis of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a median overall survival time of 5 mo and the five years survival less than 5%, a rate essentially unchanged over the course of the years. A well defined progression model of accumulation of genetic alterations ranging from single point mutations to gross chromosomal abnormalities has been introduced to describe the origin of this disease. However, due to the its subtle nature and concurring events PDAC cure remains elusive. Nuclear receptors (NR) are members of a large superfamily of evolutionarily conserved ligand-regulated DNA-binding transcription factors functionally involved in important cellular functions ranging from regulation of metabolism, to growth and development. Given the nature of their ligands, NR are very tempting drug targets and their pharmacological modulation has been widely exploited for the treatment of metabolic and inflammatory diseases. There are now clear evidences that both classical ligand-activated and orphan NR are involved in the pathogenesis of PDAC from its very early stages; nonetheless many aspects of their role are not fully understood. The purpose of this review is to highlight the striking connections that link peroxisome proliferator activated receptors, retinoic acid receptors, retinoid X receptor, androgen receptor, estrogen receptors and the orphan NR Nur, chicken ovalbumin upstream promoter transcription factor II and the liver receptor homologue-1 receptor to PDAC development, connections that could lead to the identification of novel therapies for this disease.

Axonal growth inhibitors and their receptors in spinal cord injury:from biology to clinical translation

Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.

Genomic Organization of Purinergic P2X Receptors

Purinergic P2X receptors are a family of ligand-gated cationic channels activated by extracellular ATP. P2X subunit protein sequences are highly conserved between vertebrate species. However, they can generate a great diversity of coding splicing variants to fulfill several roles in mammalian physiology. Despite intensive research in P2X expression in both central and peripheral nervous system, there is little information about their homology, genomic structure and other key features that can help to develop selective drugs or regulatory strategies of pharmacological value which are lacking today. In order to obtain clues on mammalian P2X diversity, we have performed a bioinformatics analysis of the coding regions and introns of the seven P2X subunits present in human, simian, dog, mouse, rat and zebrafish. Here we report the arrangements of exon and intron sequences, considering its number, size, phase and placement;proposing some ideas about the gain and loss of exons and retention of introns. Taken together, these evidences show traits that can be used to gain insight into the evolutionary history of vertebrate P2X receptors and better understand the diversity of subunits coding the purinergic signaling in mammals.

Electroacupuncture improves neuropathic pain Adenosine, adenosine 5'-triphosphate disodium and their receptors perhaps change simultaneously

Applying a stimulating current to acupoints through acupuncture needles–known as electroacupuncture–has the potential to produce analgesic effects in human subjects and experimental animals. When acupuncture was applied in a rat model, adenosine 5-triphosphate disodium in the extracellular space was broken down into adenosine, which in turn inhibited pain transmission by means of an adenosine A1 receptor-dependent process. Direct injection of an adenosine A1 receptor agonist enhanced the analgesic effect of acupuncture. The analgesic effect of acupuncture appears to be mediated by activation of A1 receptors located on ascending nerves. In neuropathic pain, there is upregulation of P2X purinoceptor 3 （P2X3） receptor expression in dorsal root ganglion neurons. Conversely, the onset of mechanical hyperalgesia was diminished and established hyperalgesia was significantly reversed when P2X3 receptor expression was downregulated. The pathways upon which electroacupuncture appear to act are interwoven with pain pathways, and electroacupuncture stimuli converge with impulses originating from painful areas. Electroacupuncture may act via purinergic A1 and P2X3 receptors simultaneously to induce an analgesic effect on neuropathic pain.

Role of P2X_7 receptors in neuronal death in the retina

Acknowledgments： I would like to express my appreciation to Professor Puro DG for leading me to this research topic during my stay as a research fellow in his laboratory at the University of Michigan in 2001, and also to Professor Ikeda T forgiving me the opportunity to study abroad and then to continue to investigate this topic in the Department of Ophthalmology at Osaka Medical College, lapan.

P2X receptors in maintenance and differentiation of neural progenitor cells

Purinergic receptors are among the first cell surface receptors expressed during embryonic development （Burnstock and Ulrich, 2011）. These are characterized based on their pharmacological properties of being activated by adenosine or purine/pyrimidine nucleotides as P1 and P2 receptors. P2 receptors are further classified by their structure as P2Y metabotropic and P2X ionotropic receptors.

Regulation of neural stem/progenitor cell functions by P2X and P2Y receptors

Neural stem/progenitor cells：Radial glial cells constitute multipotent cells in the ventricular zone,lining the wall of the lateral ventricle of the embryonic brain.They have the capacity to give rise to cells belonging to all three major linages（neurons,astrocytes and oligodendrocytes）of the nervous system（Tang and Illes,2017）.

P2X receptors: New players in cancer pain

Pain is unfortunately a quite common symptom for cancer patients. Normally pain starts as an episodic experience at early cancer phases to become chronic in later stages. In order to improve the quality of life of oncological patients, anti-cancer treatments are often accompanied by analgesic therapies. The P2 X receptor are adenosine triphosphate(ATP) gated ion channels expressed by several cells including neurons, cancer and immune cells. Purinergic signaling through P2 X receptors recently emerged as possible common pathway for cancer onset/growth and pain sensitivity. Indeed, tumor microenvironment is rich in extracellular ATP, which has a role in both tumor development and pain sensation. The study of the different mechanisms by which P2 X receptors favor cancer progression and relative pain, represents an interesting challenge to design integrated therapeutic strategies for oncological patients. This review summarizes recent findings linking P2 X receptors and ATP to cancer growth, progression and related pain. Special attention has been paid to the role of P2X2, P2X3, P2X4 and P2X7 in the genesisof cancer pain and to the function of P2X7 in tumor growth and metastasis. Therapeutic implications of the administration of different P2 X receptor blockers to alleviate cancer-associated pain sensations contemporarily reducing tumor progression are also discussed.

Role of P2X_7 receptors in the development of diabetic retinopathy

The P2X7 receptor is one of the members of the family of purinoceptors which are ligand-gated membrane ion channels activated by extracellular adenosine 5'-triphosphate. A unique feature of the P2X7 receptor is that its activation can result in the formation of large plasma membrane pores that allow not only the flux of ions but also of hydrophilic molecules of up to 900 Da. Recent studies indicate that P2X7-mediated signaling can trigger apoptotic cell death after ischemia and during the course of certain neurodegenerative disorders. Expression of the P2X7 receptor has been demonstrated in most types of cells in the retina. This purinoceptor mediates the contraction of pericytes and regulates the spatial and temporal dynamics of the vasomotor response through cell-to-cell electrotonic transmission within the microvascular networks. Of potential clinical significance, investigators have found that diabetes markedly boosts the vulnerability of retinal microvessels to the lethal effect of P2X7 receptor activation. This purinergic vasotoxicity may result in reduced retinal blood flow and disrupted vascular function in the diabetic retina. With recent reports indicating an association between P2X7 receptor activation and inflammatory cytokine expression in the retina, this receptor may also exacerbate the development of diabetic retinopathy by a mechanism involving inflammation.

The bumpy road of purinergic inhibitors to clinical application in immune-mediated diseases

Purinergic signaling plays important roles throughout the body in the regulation of organ functions during and following the disruption of homeostasis.This is also reflected by the widespread expression of two families of purinergic receptors(P1 and P2)with numerous subtypes.In the last few decades,there has been increasing evidence that purinergic signaling plays an important role in the regulation of immune functions.Mainly,signals mediated by P2 receptors have been shown to contribute to immune system-mediated pathologies.Thus,interference with P2 receptors may be a promising strategy for the modulation of immune responses.Although only a few clinical studies have been conducted in isolated entities with limited success,preclinical work suggests that the use of P2 receptor inhibitors may bear some promise in various autoimmune diseases.Despite the association of P2 receptors with several disorders from this field,the use of P2 receptor antagonists in clinical therapy is still very scarce.In this narrative review,we briefly review the involvement of the purinergic system in immunological responses and clinical studies on the effect of purinergic inhibition on autoimmune processes.We then open the aperture a bit and show some preclinical studies demonstrating a potential effect of purinergic blockade on autoimmune events.Using suramin,a non-specific purinergic inhibitor,as an example,we further show that off-target effects could be responsible for observed effects in immunological settings,which may have interesting implications.Overall,we believe that it is worthwhile to further investigate this hitherto underexplored area.

Regulatory effect of mild moxibustion on P2X3 receptors in spinal cord,anterior cingulate cortex and thalamic ventral posterolateral nucleus of rats with IBS visceral hyperalgesia

Objective To observe the therapeutic effect of mild moxibustion on irritable bowel syndrome(IBS)visceral hyperalgesia model rats and its regulatory effect on P2X3 receptors in the spinal cord,anterior cingutate cortex(ACC)and thalamic ventral posterolateral nucleus(VPL).Methods Thirty 8-day-old newborn rats were randomly divided into a normal group(n=6)and a modeling group(n=24)according to the completely random number table method.Rats in the normal group were bred routinely,and those in the modeling group were subjected to preparing IBS chronic visceral hyperalgesia model using colorectal distention(CRD)in stimulation method.Rats successfully modelled were re-divided into a model group,a mild moxibustion group,a P2X3 receptor antagonist group,and a normal saline group according to the completely random number table method with 6 rats in each group.Rats in each group received corresponding interventions from the 37-day old,once a day for 7 consecutive days.Immunohistochemistry and Western blot assays were used to detect P2X3 protein expressions in the spinal cord,ACC and VPL of rats.Results Under different intensities of CRD stimulation,the abdominal withdrawal reflex(AWR)scores of the model group were significantly increased versus the normal group(all P<0.05);the AWR scores of the mild moxibustion group and the P2X3 receptor antagonist group were significantly reduced versus the model group(all P<0.01).The P2X3 protein expressions in rat spinal cord,ACC and VPL tissues of the model group were significantly increased versus the normal group(all P<0.01);the P2X3 protein expressions in rat spinal cord,ACC and VPL tissues of the mild moxibustion group and the P2X3 receptor antagonist group were significantly reduced versus the model group(all P<0.01).Conclusion Mild moxibustion can inhibit the P2X3 receptor expressions in the spinal cord,ACC,and VPL tissues of IBS visceral hyperalgesia model rats,which may be the mechanism of mild moxibustion in relieving the central sensitization of rats with IBS visceral hyperalgesia.

Molecular mechanism underlying the subtype-selectivity of competitive inhibitor NF110 and its distinct potencies in human and rat P2X3 receptors

P2X receptors are a family of extracellular ATP-gated trimeric cation channels that is widely distributed in human tissues. Quite some drug candidates targeting P2X receptors have entered into preclinical or main phases of clinical trials, but many of them failed due to low subtype-selectivity or species differences in pharmacological activities between human and experimental animals. Here, we identified the distinct inhibitory efficacies of NF110, a competitive inhibitor, between the rat(rP2X3) and human(hP2X3) P2X3 receptors. We demonstrated that this difference is determined by two amino acids located in the dorsal fin(DF) domain of P2X3 receptors. As revealed by mutagenesis, metadynamics, and covalent modification, NF110-mediated rP2X3 inhibition may be through a filling in the cavity formed by the DF,left flipper(LF) and lower body(LB) to partially, rather than fully, occupy the ATP-binding pocket.Moreover, substitution of residues located in the DF and/or LF domains of the rP2X2 receptor, a NF110-insensitive subtype, with the equivalent amino acids of rP2X3, bestowed the sensitivity of rP2X2 to NF110. The critical roles of the DF and LF domains in channel gating of P2X and low conservativity in residue sequences of those two domains raise the possibility that small molecules differentially interacting with the residues of the DF and LF domains of different P2X receptors may modulate channel's activity in a subtype-selective manner. However, the possible species-specificity of P2X inhibitors/modulators makes it more complex when interpreting the preclinical data into clinical researches.Nevertheless, our data provide new insights into the subtype-selectivity of competitive inhibitors and their distinct potencies in the human and experimental animals, both of which are extremely important in the drug discovery of P2X receptors.

Overexpression of Purinergic P2X4 Receptors in Hippocampus Rescues Memory Impairment in Rats with Type 2 Diabetes

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(T2 DM).T2 DM rats exhibited a worse performance in the T-maze and Morris water maze(MWM) than controls.Microglia positive for P2 X purinoceptor 4(P2 X4 R) in the hippocampus were reduced and activated microglia were increased in T2 DM rats.Long Amplicon PCR(LA-PCR) showed that DNA amplification of the p2 x4 r gene in the hippocampus was lower in T2 DM rats.Minocycline significantly reduced the number of activated microglia and the mean distance traveled by T2 DM rats in the MWM.Most importantly,P2 X4 R overexpression suppressed the activated microglia and rescued the memory impairment of T2 DM rats.Overall,T2 DM led to excessive activation of microglia in the hippocampus,partly through the DNA damagemediated downregulation of P2 X4 Rs,thus contributing to memory impairment.

Triggering of Major Brain Disorders by Protons and ATP:The Role of ASICs and P2X Receptors

Adenosine triphosphate(ATP)is well-known as a universal source of energy in living cells.Less known is that this molecule has a variety of important signaling func-tions:it activates a variety of specific metabotropic(P2Y)and ionotropic(P2X)receptors in neuronal and non-neu-ronal cell membranes.So,a wide variety of signaling func-tions well fits the ubiquitous presence of ATP in the tissues.Even more ubiquitous are protons.Apart from the unspe-cific interaction of protons with any protein,many physi-ological processes are affected by protons acting on specific ionotropic receptors--acid-sensing ion channels(ASICs).Both protons(acidification)and ATP are locally elevated in various pathological states.Using these fundamentally important molecules as agonists,ASICs and P2X receptors signal a variety of major brain pathologies.Here we briefly outline the physiological roles of ASICs and P2X receptors,focusing on the brain pathologies involving these receptors.

Gefapixant,a Novel P2X3 Antagonist,Protects against Post Myocardial Infarction Cardiac Dysfunction and Remodeling Via Suppressing NLRP3 Inflammasome

Objective The ATP responsive P2 purinergic receptors can be subdivided into metabotropic P2X family and ionotropic P2Y family.Among these,P2X3 is a type of P2X receptor which is specifically expressed on nerves,especially on pre-ganglionic sensory fibers.This study investigates whether gefapixant possesses the potential of inhibiting cardiac sympathetic hypersensitivity to protect against cardiac remodeling in the context of myocardial infarction.Methods The Sprague-Dawley rats were divided randomly into three groups:sham group-myocardial infarction group,and myocardial infarction with gefapixant treatment group.Myocardial infarction was induced by left anterior descending branch ligation.The gefapixant solution was intraperitoneally injected each time per day for 7 days and the appropriate dosage of gefapixant was determined according to the results of hematoxylin-eosin(HE)staining and myocardial injury biomarkers.Conditions of cardiac function were assessed by echocardiograph and cardiac fibrosis was evaluated by Western blotting and immunofluorescence staining of collagen I and collagen III.The sympathetic innervation was detected by norepinephrine concentration(pg/mL),in-vivo electrophysiology,and typical sympathetic biomarkers.Inflammatory cell infiltration was shown from immunofluorescence staining and pro-inflammatory signaling pathway activation was checked by immunohistology,quantitative realtime PCR(qPCR)and Western blotting.Results It was found that gefapixant injection of 10 mg/kg per day had the highest dosage-efficacy ratio.Furthermore,gefapixant treatment improved cardiac pump function as shown by increased LVEF and LVFS,and decreased LVIDd and LVIDs.The expression levels of collagen I and collagen III,and TNF-αwere all decreased by P2X3 inhibition.Mechanistically,the decreased activation of nucleotide-binding and oligomerization domain-like receptors family pyrin-domain-containing 3(NLRP3)inflammasome and subsequent cleavage of caspase-1 which modulated interleukin-1β(IL-1β)and IL-18 level in heart after gefapixant treatment were associated with the suppressed cardiac inflammation.Conclusion It is suggested that P2X3 inhibition by gefapixant ameliorates post-infarct autonomic nervous imbalance,cardiac dysfunction,and remodeling possibly via inactivating NLRP3 inflammasome.

背根神经节嘌呤受体亚型P2X3R介导小鼠术后急—慢痛转化

目的探讨不同部位不同嘌呤受体亚型在小鼠切口术后痛转化中的作用差异。方法足底切口恢复后注射PGE_(2)构建术后痛转化模型。第一部分:C57BL/6小鼠随机分为对照(Con)组、假敏化(sHP)组、敏化(HP)组,检测机械痛阈、自发痛评分和焦虑样行为;Western blot检测患侧L 3-5背根神经节(DRG)和脊髓背角(SCDH)中P2X3R和P2X7R蛋白表达变化。第二部分:C57BL/6小鼠随机分为Con组、HP+生理盐水组、HP+P2X3R拮抗剂组或HP+P2X7R拮抗剂组,观察拮抗不同亚型嘌呤受体对小鼠机械痛阈的影响。结果与正常小鼠相比,切口痛小鼠注射PGE_(2)后机械痛阈明显下降,自发痛评分明显增加(P<0.05),未表现出焦虑样行为(P>0.05),且P2X3R蛋白在DRG中表达明显增多(P<0.05),在SCDH中未检出,而P2X7R蛋白在DRG和SCDH各组中表达均无差异(P>0.05)。拮抗P2X3R能阻断术后痛转化的产生,而拮抗P2X7R无明显影响。结论切口痛小鼠经PGE_(2)诱导可出现急慢性痛的转化,不伴发焦虑样情绪,DRG中P2X3R可能是参与术后痛转化的关键受体之一。

Pulsed radiofrequency inhibits expression of P2X3 receptors and alleviates neuropathic pain induced by chronic constriction injury in rats

Background:Pulsed radiofrequency (PRF) is a minimally invasive interventional technique that provides a novel and effective treatment strategy for neuropathic pain (NP).PRF is advantageous because it does not damage nerves and avoids sensory loss after treatment.At present,animal studies have demonstrated that PRF is safe and effective for relieving the NP associated with sciatic nerve damage in rats with chronic constriction injury (CCI).However,the mechanism through which this effect occurs is unknown.An increasing body of evidence shows that the expression of the P2X ligand-gated ion channel 3 (P2X3) receptor is closely related to NP;this study was to investigate whether the expression of this receptor is involved in NP relief due to PRF.Methods:A total of 36 healthy adult male Sprague-Dawley (SD) rats were randomly divided into three groups:Sham group,CCI group,and PRF group.The right sciatic nerve was ligated in CCI group and PRF group to establish a CCI model;the right sciatic nerve was separated but not ligated in Sham group.On day 14 after the operation,PRF was administered to the ligated sciatic nerve in PRF group (42℃,45 V,2 min).A non-live electrode was placed at the exposed sciatic nerve for the rats in Sham and CCI groups.The hindpaw withdrawal threshold (HWT) and thermal withdrawal latency (TWL) were measured at the right hindpaw at different time points before and after PRF or sham therapy.On day 28 after treatment,the dorsal root ganglion (DRG) and spinal dorsal horn of the right L4-6 were harvested from each group to determine the mRNA and protein levels of the P2X3 receptor.Results:On day 28 after PRF treatment,the HWT (8.33 ± 0.67 g vs.3.62 ± 0.48 g) and TWL (25.42 ± 1.90 s vs.15.10 ± 1.71 s) were significantly higher in PRF group as compared to CCI group (P < 0.05).The mRNA expression of the P2X3 receptor in the DRG in PRF group was 23.7% lower than that in CCI group (P < 0.05),in the spinal dorsal horns in PRF group was 22.7% lower than that in CCI group (P < 0.05).The protein expression of the P2X3 receptor in the DRG in PRF group was 27.8 % lower than that in CCI group (P < 0.05),in the spinal dorsal horns in PRF group was 35.6% lower than that in CCI group (P < 0.05).Conclusion:PRF possibly reduces NP in CCI rats by inhibiting the expression of the P2X3 receptor in the L4-6 DRG and spinal dorsal horns.

P2X7R过表达的巨噬细胞MSU晶体诱导痛风炎症反应过程中IL-1β、TNF-α、NLRP3表达观察

目的观察嘌呤能受体P2X配体门控离子通道7的配体(P2X7R)过表达白血病细胞诱导分化的巨噬细胞单钠尿酸盐(MSU)晶体诱导痛风炎症反应过程中NOD样受体家族3(NLRP3)蛋白、IL-1β、TNF-α表达情况。方法取人单核细胞白血病细胞系THP-1,并随机分为过表达组、空白组、模型组、对照组;过表达组和空白组分别转染P2X7R过表达质粒、空白载体质粒,转染5 d,将过表达组、空白组、模型组THP-1细胞用100 ng/mL的PMA刺激3 h后分化为巨噬细胞,另将MSU晶体用氢氧化钠溶解配制成浓度为100μg/mL的MSU乳糜状悬液加入培养液中孵育6 h;对照组正常培养。分别采用RT-PCR法和Western blot法测算巨噬细胞P2X7R mRNA、蛋白,ELISA法检测巨噬细胞上清液IL-1β、TNF-α,Western blot法测算巨噬细胞NOD样受体家族3(NLRP3)蛋白。结果与对照组比较,过表达组、空白组、模型组P2X7R mRNA和蛋白相对表达量升高,细胞上清液IL-1β、TNF-α水平升高,细胞NLRP3蛋白相对表达量升高(P均<0.05);与模型组、空白组比较,过表达组P2X7R mRNA、蛋白相对表达量升高,细胞上清液IL-1β、TNF-α水平升高,细胞NLRP3蛋白相对表达量升高(P均<0.05)。结论P2X7R过表达白血病细胞诱导分化的巨噬细胞MSU晶体诱导痛风炎症反应过程中IL-1β、TNF-α、NLRP3表达增加,IL-1β、TNF-α水平升高可能通过激活NLRP3蛋白来实现。

槲皮素缓解紫杉醇诱发外周神经病变小鼠模型的疼痛效应及其机制研究

目的观察槲皮素对紫杉醇诱发外周神经病变小鼠模型疼痛症状、脊髓星形胶质细胞活化以及背根神经节中痛敏感瞬时感受器电位香草酸亚型1(transient receptor potential vanilloid 1,TRPV1)、嘌呤能配体门控离子通路3(purinergic ligand-gated ion channel 3,P2X3)的干预作用。方法选取C57BL/6小鼠24只,随机分为空白组、模型组、模型+槲皮素组,每组8只。模型组与模型+槲皮素组分别注射累计剂量为8 mg/kg的紫杉醇进行造模,空白组在相应时间点注射对应溶剂。模型+槲皮素组在造模第8天开始,每天灌胃60 mg/kg的槲皮素溶液,其余两组分别给予相应体积溶剂。纤毛机械刺激针(von Frey)检测各组小鼠的机械痛痛阈,免疫荧光检测各组小鼠脊髓中星形胶质细胞的活化,免疫荧光和免疫印迹检测背根神经节中痛敏感TRPV1、P2X3的表达。结果(1)与模型组相比,模型+槲皮素组小鼠的机械痛痛阈显著升高,疼痛症状得到明显缓解。(2)与模型组相比,模型+槲皮素组小鼠脊髓星形胶质细胞的活化明显降低,背根神经节中TRPV1、P2X3的表达显著减少(P<0.05)。结论槲皮素能够有效缓解紫杉醇诱发外周神经病变小鼠的疼痛症状,其作用机制很可能与缓解脊髓星形胶质细胞活化以及减少背根神经节中TRPV1、P2X3表达有关。