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.

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.

Purinergic P2Y Receptors Are Involved in Xenopus Head Formation

P2Y receptors belong to the family of G protein-coupled receptors and are activated by nucleotides in the extracellular space. We showed that Xenopus P2Y1 and P2Y11 were expressed in the dorsal marginal zone from early gastrula stage and enriched in the central nervous system from neurula stages. They were expressed in the prospective head region during early development. Knockdown of P2Y1 and P2Y11 caused head malformation, such as small eyes, brain atrophy, and defect in cartilage tissues, as well as reduced expression of neural, placode, and neural crest markers. Furthermore, the expression of neural plate and epidermal markers was affected by P2Y1 or P2Y11 depletion at early neurula stage, suggesting that P2Y1 or P2Y11 might be required for the neural induction. Our findings suggested that P2Y receptors might be involved in distinguishing between neural and non-neural fates. The results also suggested that P2Y1 or P2Y11 could play a role in neural induction and/or maintenance of neural tissues in the head formation processes.

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.

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）.

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.

P2Y1 receptor in Alzheimer’s disease

Alzheimer’s disease is the most frequent form of dementia characterized by the deposition of amyloid-beta plaques and neurofibrillary tangles consisting of hyperphosphorylated tau.Targeting amyloid-beta plaques has been a primary direction for developing Alzheimer’s disease treatments in the last decades.However,existing drugs targeting amyloid-beta plaques have not fully yielded the expected results in the clinic,necessitating the exploration of alternative therapeutic strategies.Increasing evidence unravels that astrocyte morphology and function alter in the brain of Alzheimer’s disease patients,with dysregulated astrocytic purinergic receptors,particularly the P2Y1 receptor,all of which constitute the pathophysiology of Alzheimer’s disease.These receptors are not only crucial for maintaining normal astrocyte function but are also highly implicated in neuroinflammation in Alzheimer’s disease.This review delves into recent insights into the association between P2Y1 receptor and Alzheimer’s disease to underscore the potential neuroprotective role of P2Y1 receptor in Alzheimer’s disease by mitigating neuroinflammation,thus offering promising avenues for developing drugs for Alzheimer’s disease and potentially contributing to the development of more effective treatments.

P2Y1受体介导星形胶质细胞的激活在急性一氧化碳中毒迟发性脑病中的作用

目的探讨P2Y1受体和星形胶质细胞在急性一氧化碳(CO)中毒迟发性脑病(DEACMP)中的作用以及DEACMP可能的发病机制。方法经水迷宫实验筛选认知功能合格的雄性SD大鼠,随机分为两组:对照组、CO中毒组,CO中毒组制作DEACMP模型,分别于造模后第7天、第14天、第21天、第28天对比两组行为学改变,神经元变化以及海马组织中P2Y1受体和星型胶质细胞的表达。结果通过水迷宫发现与对照组相比,造模后第21天、第28天CO中毒组大鼠逃避潜伏期均明显延长(P<0.05);HE染色发现造模后第14天、第21天、第28天模型组大鼠海马锥体细胞及神经元坏死明显,结合水迷宫可表明大鼠在21 d时出现DEACMP;与对照组相比,Western blot法检测提示各时间点CO中毒组海马区P2Y1及GFAP蛋白表达均增多(P<0.05),呈先升高后降低的趋势;免疫荧光表明海马区P2Y1和GFAP存在共表达,相对于对照组,中毒后各时间点海马CA 1区P2Y1和GFAP都有表达上调(P<0.05)。结论P2Y1受体对星形胶质细胞的激活可能是DEACMP的发病机制之一,星形胶质细胞可能通过介导免疫炎症反应使CO中毒的大鼠学习记忆能力损害导致DEACMP发生。

Prolonged intermittent theta burst stimulation restores the balance between A_(2A)R-and A_(1)R-mediated adenosine signaling in the 6-hydroxidopamine model of Parkinson's disease

An imbalance in adenosine-mediated signaling,particularly the increased A_(2A)R-mediated signaling,plays a role in the pathogenesis of Parkinson's disease.Existing therapeutic approaches fail to alter disease progression,demonstrating the need for novel approaches in PD.Repetitive transcranial magnetic stimulation is a non-invasive approach that has been shown to improve motor and non-motor symptoms of Parkinson's disease.However,the underlying mechanisms of the beneficial effects of repetitive transcranial magnetic stimulation remain unknown.The purpose of this study is to investigate the extent to which the beneficial effects of prolonged intermittent theta burst stimulation in the 6-hydroxydopamine model of experimental parkinsonism are based on modulation of adenosine-mediated signaling.Animals with unilateral 6-hydroxydopamine lesions underwent intermittent theta burst stimulation for 3 weeks and were tested for motor skills using the Rotarod test.Immunoblot,quantitative reverse transcription polymerase chain reaction,immunohistochemistry,and biochemical analysis of components of adenosine-mediated signaling were performed on the synaptosomal fraction of the lesioned caudate putamen.Prolonged intermittent theta burst stimulation improved motor symptoms in 6-hydroxydopamine-lesioned animals.A 6-hydroxydopamine lesion resulted in progressive loss of dopaminergic neurons in the caudate putamen.Treatment with intermittent theta burst stimulation began 7 days after the lesion,coinciding with the onset of motor symptoms.After treatment with prolonged intermittent theta burst stimulation,complete motor recovery was observed.This improvement was accompanied by downregulation of the e N/CD73-A_(2A)R pathway and a return to physiological levels of A_(1)R-adenosine deaminase 1 after 3 weeks of intermittent theta burst stimulation.Our results demonstrated that 6-hydroxydopamine-induced degeneration reduced the expression of A_(1)R and elevated the expression of A_(2A)R.Intermittent theta burst stimulation reversed these effects by restoring the abundances of A_(1)R and A_(2A)R to control levels.The shift in ARs expression likely restored the balance between dopamine-adenosine signaling,ultimately leading to the recovery of motor control.

Electroacupuncture diminishes P2X_2 and P2X_3 purinergic receptor expression in dorsal root ganglia of rats with visceral hypersensitivity

Electroacupuncture at Shangjuxu （ST37） and Tianshu （ST25） can improve visceral hypersensitivity in rats. Colorectal distension was used to establish a rat model of chronic visceral hypersensitivity. Immunohistochemistry was used to detect P2X2 and P2X3 receptor expression in dorsal root ganglia from rats with chronic visceral hypersensitivity. Results demonstrated that abdominal withdrawal reflex scores obviously increased following establishment of the model, indicating visceral hypersensitivity. Simultaneously, P2X2 and P2X3 receptor expression increased in dorsal root ganglia. After bilateral electroacupuncture at Shangjuxu and Tianshu, abdominal withdrawal reflex scores and P2X2 and P2X3 receptor expression decreased in rats with visceral hypersensitivity. These results indicated that electroacupuncture treatment improved visceral hypersensitivity in rats with irritable bowel syndrome by reducing P2X2 and P2X3 receptor expression in dorsal root ganglia.

三叉神经痛模型大鼠三叉神经节中P2Y1受体的时序表达分析

目的:探讨P2Y1受体在三叉神经痛大鼠三叉神经节神经元内的时序表达。方法:雄性SD大鼠68只,随机分成3组。正常对照组(4只)未做任何处理;假手术组(32只)暴露眶下神经但不做结扎;手术组(32只)暴露眶下神经并结扎右侧。假手术组和手术组按照术后各时间点(每时间点4只)取出三叉神经节。行为学观察、HE染色评价动物模型,免疫组化检测并分析TG中P2Y1受体的表达情况。结果:正常对照组中P2Y1受体在TG神经元中表达较弱。手术侧各时间点P2Y1受体的表达均增加。手术侧与假手术组各时间点的P2Y1受体表达量变化存在差异。手术侧P2Y1受体的表达先增加后逐渐回落,手术侧该受体各时间点的表达量均高于假手术组。手术侧与手术对侧各时间点的P2Y1受体表达量变化差异较小。结论:P2Y1受体在CCI-ION模型大鼠的TG神经元内的表达出现不同程度的波动,可能在神经痛中具有一定的作用,且手术侧的痛觉神经传导对对侧TG产生了影响。

Purinergic contraction of the rat vas deferens in L-NAME-induced hypertension：effect of sildenafil

Hypertension （HTN） is a risk factor for erectile dysfunction, but its effect on vas deferens （VD） contractility and the ejaculatory response has not been delineated. NG-nitro-L-arginine methyl ester （L-NAME）, a nitric oxide synthase inhibitor, was used for induction of nitric oxide （NO）-deficient HTN. Our aim was to evaluate the effects of L-NAME-induced HTN on rat VD contractility and to determine whether sildenafil affects VD contractility. A total of 36 male rats were divided into （1） control, （2）L-NAME-HTN, （3） sildenafil treated L-NAME-HTN groups. Group 2 was treated with L-NAME （40 mg kgI per day） in drinking water for 4 weeks. Group 3 received sildenafil （1.5 mg kg^-1 per day, by oral gavage） concomitantly with L-NAME. The prostatic portion of the VD was subjected to electrical field stimulation （EFS, 1-20 Hz）, and the P2X1 agonist α,β-methylene ATP （α,β meATP, 100 μmol L^-1-1 μmol L-1） and the al-adrenoceptor agonist phenylephrine （Phe, 100 μmol L^-1-1 mmol L^-1） were used to construct concentration-response curves. These experiments were repeated in the presence of P2X receptor antagonist, pyridoxalphosphate-6-azophenyl-2＇,4＇-disulfonic acid （PPADS, 30 μmol L-1）. VD contractions in response to EFS, a,β-meATP and Phe were significantly enhanced by L-NAME. Sildenafil treatment in the L-NAME group improved the contractile response of VD to EFS （20 Hz）. In the presence of PPADS, the enhanced contractile response of VD to EFS and a,β-meATP in hypertensive rats was reversed. In the rat model of chronic NO depletion, the purinergic and adrenergic components and EFS affect VD contractility. The VD contractile response may be mediated more by the purinergic system than the adrenergic system, and sildenafil may alter the ejaculatory response in men with PE.

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.

P2Y1R is involved in visceral hypersensitivity in rats with experimental irritable bowel syndrome

AIM To evaluate the role of P2Y1 R in visceral hypersensitivity in rats with experimental irritable bowel syndrome.METHODS A rat model of irritable bowel syndrome was generated by intra-colonic administration of acetic acid(AA) and assessed by histology and myeloperoxidase(m PO) activity assay. Then P2Y1 R expression in the colonic tissue was detected by Western blot. In order to explore the regulatory role of P2Y1 R in visceral hypersensitivity, an agonist(m RS2365) and an antagonist(m RS2179) of P2Y1 R were intra-colonically administered and effects were tested through a colorectal distension test. The abdominal withdrawal reflex and abdominal electromyography were tested during the course. RESULTS model assessment tests showed an obvious inflammatoryreaction that appeared on the 2^(nd) d after the AA injection, and the inflammatory reaction gradually recovered and almost disappeared on the 7^(th) d. The model finished on day 8 and showed a clear feature of IBS that had no organic lesion. The average expression of P2Y1 R was significantly higher in the AA group than in the na?ve group(0.319 ± 0.02 vs 0.094 ± 0.016, P < 0.001). m RS2365 could effectively raise the colonic hypersensitivity status at intervention doses of 10(AUC value from 0.30 ± 0.089 to 1.973 ± 0.127 mv?s, P < 0.01) and 100 μmol/L(AUC value from 0.290 ± 0.079 to 1.983 ± 0.195 mv?s, P < 0.01); m RS2179 could effectively reduce the hypersensitivity status at intervention dose of 100 μmol/L(from a mean baseline AUC value of 1.587 ± 0.099 mv?s to 0.140 ± 0.089 mv?s, P < 0.0001). Differences between the m RS2179 group(1.88 ± 1.45) and either the m RS2365 group(3.96 ± 0.19) or the combined treatment(m RS2179 and m RS2365) group(3.28 ± 0.11) were significant(P < 0.01).CONCLUSION P2Y1 R plays a regulatory role in visceral hypersensitivity in rats with experimental IBS. Specific antagonists of P2Y1 R may have potential therapeutic value in treating abdominal pain in IBS.

Up-regulation of P2X7 Receptors Contributes to Spinal Microglial Activation and the Development of Pain Induced by BmK-I

Previous work has demonstrated that the sensitization of spinal neurons and microglia is important in the development of pain behaviors induced by BmK I,a Na^+ channel activator and a major peptide component of the venom of the scorpion Buthus martensi Karsch(BmK).We found that the expression of P2X7 receptors(P2X7Rs)was up-regulated in the ipsilateral spinal dorsal horn after BmK I injection in rats.P2X7R was selectively localized in microglia but not astrocytes or neurons.Similarly,interleukin 1β(IL-1β)was selectively up-regulated in microglia in the spinal dorsal horn after BmK I injection.Intrathecal injection of P2X7R antagonists largely reduced BmK I-induced spontaneous and evoked pain behaviors,and the up-regulation of P2X7R and IL-1β in the spinal cord.These data suggested that the up-regulation of P2X7Rs mediates microglial activation in the spinal dorsal horn,and therefore contributes to the development of BmK I-induced pain.

大黄䗪虫丸中基于血小板P2Y1及P2Y12受体作用的抗血小板聚集活性成分筛选

目的结合分子对接和血小板聚集实验对大黄䗪虫丸中基于血小板P2Y1及P2Y12受体作用的抗血小板聚集活性成分进行初步筛选。方法以P2Y1及P2Y12受体作为靶受体,分别与大黄䗪虫丸中9种主要成分(大黄酚、大黄素、大黄酸、大黄素甲醚、芦荟大黄素、黄芩苷、苦杏仁苷、芍药苷和甘草酸)进行分子对接,计算结合能并分析分子间作用力,与P2Y1及P2Y12受体自带活性配体进行比较。随后以腺苷-5’-二磷酸钠(ADP)为诱导剂,观察大黄䗪虫丸中9种主要成分在同一浓度下对血小板聚集的作用,以此初步验证分子对接的结果。结果分子对接结果显示,甘草酸、黄芩苷和大黄酸与P2Y1受体结合能较低,结合位置与自带配体较相似;大黄酸、大黄酚、大黄素、大黄素甲醚和芦荟大黄素与P2Y12受体结合能相对较低,结合位置与自带配体相对较相似。ADP诱导的血小板聚集实验中,2 nmol·L^(-1)的大黄酸和芦荟大黄素与空白组相比具有显著的抑制作用,聚集率分别为41.3%和73.1%,抑制率为51.2%和13.5%。结论结合分子对接和血小板聚集实验结果,推测大黄酸和芦荟大黄素可能是大黄䗪虫丸中基于P2Y1及P2Y12受体通路的抑制血小板聚集的主要活性成分。

Ghosts in the shell:identification of microglia in the human central nervous system by P2Y12 receptor

Microglia are the tissue resident macrophages of the brain and represent the sole immune population located in the parenchyma of the central nervous system （CNS）. These cells are hidden be-tween neurons, astrocytes as well as oligodendrocytes and account for only 5-10% of CNS cells. Even though microglia were already identified in 1913 by the Spanish neuroanatomist Ramon y Cajal and further seminally investigated by his student Pio del Rio Hortega,

MicroRNA-133b调节FGFR1-ERK1/2-SOX2信号通路对裸鼠肺癌NCI-H1975细胞移植瘤生长的影响

目的探讨microRNA-133b(miR-133b)对裸鼠肺癌NCI-H1975细胞移植瘤生长的抑制作用以及对成纤维细胞生长因子受体1-细胞外信号调节激酶1/2-性别决定区Y-box蛋白2信号通路(FGFR1-ERK1/2-SOX2)的影响。方法q RT-PCR检测人肺成纤维细胞、肺癌细胞株miR-133b表达。miR-133b过表达NCIH1975细胞。将NCI-H1975细胞分为对照组、mimic NC组、miR-133b mimic组、miR-133b mimic+pcDNA3.1组、miR-133b mimic+pcDNA3.1 FGFR1组。CCK-8法检测NCI-H1975细胞增殖抑制率,Transwell实验观察NCI-H1975细胞侵袭、迁移情况。复制裸鼠移植瘤模型并分组,将裸鼠分为对照组、mimic NC组、miR-133b mimic组、miR-133b mimic+AZD4547组,观察各组裸鼠肿瘤体积与重量,HE染色观察各组裸鼠肿瘤组织变化,TUNEL检测肿瘤组织细胞凋亡情况,免疫组织化学法观察裸鼠肿瘤组织Ki-67、Cyclin D1、VEGF-A的表达,Western blotting检测各组肿瘤组织FGFR1、p-ERK1/2/ERK1/2、SOX2蛋白相对表达量。结果与人肺成纤维细胞HLF-α比较,肺癌细胞株NCI-H1975、A427、NGE-1、A549中miR-133b mRNA相对表达量降低(P<0.05),其中以NCI-H1975细胞中miR-133b mRNA相对表达量最低。miR-133b mimic组miR-133b mRNA相对表达量较对照组和mimic NC组升高(P<0.05)。miR-133b可通过负调控FGFR1抑制肺癌NCIH1975细胞增殖和迁移。miR-133b mimic组移植瘤重量较对照组降低、体积缩小,miR-133b mimic+AZD4547组移植瘤重量较miR-133b mimic组降低、体积缩小(P<0.05)。miR-133b mimic组空泡样变性程度较对照组、mimic NC组减轻(P<0.05),miR-133b mimic+AZD4547组空泡样变性程度较miR-133b mimic组减轻(P<0.05)。miR-133b mimic组肿瘤组织细胞凋亡率较对照组升高(P<0.05),miR-133b mimic+AZD4547组肿瘤组织细胞凋亡率较miR-133b mimic组升高(P<0.05)。miR-133b mimic组VEGF-A、Cyclin D、Ki-67阳性细胞比例较对照组降低(P<0.05),miR-133b mimic+AZD4547组VEGF-A、Cyclin D、Ki-67阳性细胞比例较miR-133b mimic组降低(P<0.05)。miR-133b mimic组FGFR1、p-ERK1/2/ERK1/2、SOX2蛋白相对表达量较对照组降低(P<0.05),miR-133b mimic+AZD4547组FGFR1、p-ERK1/2/ERK1/2、SOX2蛋白相对表达量较miR-133b mimic组降低(P<0.05)。结论miR-133b过表达可能通过抑制FGFR1-ERK1/2-SOX2轴,抑制裸鼠肺癌NCI-H1975细胞移植瘤生长。

医用臭氧缓解神经病理性疼痛的作用机制

目的:探讨医用臭氧(OZ)缓解神经病理性疼痛(NP)的作用机制。方法:将100只成年雄性SD大鼠随机分为空白组、手术组、低剂量组、中剂量组、高剂量组,各20只。除空白组外,其余组大鼠以手术方法构建坐骨神经慢性压迫性损伤(CCI)模型。低剂量组、中剂量组、高剂量组分别注射低、中、高剂量OZ到损伤的神经周围。比较五组机械痛阈值(MWT)、中脑导水管周围灰质外侧区P2Y1受体表达情况。结果:实验第1天,手术组MWT低于空白组,低剂量组、中剂量组、高剂量组低于空白组,但高于手术组,差异有统计学意义(P<0.05);实验第3、5、7天,高剂量组、中剂量组、低剂量组MWT逐渐升高,且空白组>高剂量组>中剂量组>低剂量组>手术组,差异有统计学意义(P<0.05)。P2Y1受体表达量高剂量组>中剂量组>低剂量组>手术组>空白组,差异有统计学意义(P<0.05)。结论:OZ对NP大鼠有明显镇痛作用,可升高大鼠MWT,增加P2Y1受体表达量。

Study of the roles of caspase-3 and nuclear factor kappa B in myenteric neurons in a P2X7 receptor knockout mouse model of ulcerative colitis

BACKGROUND The literature indicates that the enteric nervous system is affected in inflammatory bowel diseases(IBDs)and that the P2X7 receptor triggers neuronal death.However,the mechanism by which enteric neurons are lost in IBDs is unknown.AIM To study the role of the caspase-3 and nuclear factor kappa B(NF-κB)pathways in myenteric neurons in a P2X7 receptor knockout(KO)mouse model of IBDs.METHODS Forty male wild-type(WT)C57BL/6 and P2X7 receptor KO mice were euthanized 24 h or 4 d after colitis induction by 2,4,6-trinitrobenzene sulfonic acid(colitis group).Mice in the sham groups were injected with vehicle.The mice were divided into eight groups(n=5):The WT sham 24 h and 4 d groups,the WT colitis 24 h and 4 d groups,the KO sham 24 h and 4 d groups,and the KO colitis 24 h and 4 d groups.The disease activity index(DAI)was analyzed,the distal colon was collected for immunohistochemistry analyses,and immunofluorescence was performed to identify neurons immunoreactive(ir)for calretinin,P2X7 receptor,cleaved caspase-3,total caspase-3,phospho-NF-κB,and total NF-κB.We analyzed the number of calretinin-ir and P2X7 receptor-ir neurons per ganglion,the neuronal profile area(μm^(2)),and corrected total cell fluorescence(CTCF).RESULTS Cells double labeled for calretinin and P2X7 receptor,cleaved caspase-3,total caspase-3,phospho-NF-κB,or total NF-κB were observed in the WT colitis 24 h and 4 d groups.The number of calretinin-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups,respectively(2.10±0.13 vs 3.33±0.17,P<0.001;2.92±0.12 vs 3.70±0.11,P<0.05),but was not significantly different between the KO groups.The calretinin-ir neuronal profile area was increased in the WT colitis 24 h group compared to the WT sham 24 h group(312.60±7.85 vs 278.41±6.65,P<0.05),and the nuclear profile area was decreased in the WT colitis 4 d group compared to the WT sham 4 d group(104.63±2.49 vs 117.41±1.14,P<0.01).The number of P2X7 receptor-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups,respectively(19.49±0.35 vs 22.21±0.18,P<0.001;20.35±0.14 vs 22.75±0.51,P<0.001),and no P2X7 receptor-ir neurons were observed in the KO groups.Myenteric neurons showed ultrastructural changes in the WT colitis 24 h and 4 d groups and in the KO colitis 24 h group.The cleaved caspase-3 CTCF was increased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups,respectively(485949±14140 vs 371371±16426,P<0.001;480381±11336 vs 378365±4053,P<0.001),but was not significantly different between the KO groups.The total caspase-3 CTCF,phospho-NF-κB CTCF,and total NF-κB CTCF were not significantly different among the groups.The DAI was recovered in the KO groups.Furthermore,we demonstrated that the absence of the P2X7 receptor attenuated inflammatory infiltration,tissue damage,collagen deposition,and the decrease in the number of goblet cells in the distal colon.CONCLUSION Ulcerative colitis affects myenteric neurons in WT mice but has a weaker effect in P2X7 receptor KO mice,and neuronal death may be associated with P2X7 receptor-mediated caspase-3 activation.The P2X7 receptor can be a therapeutic target for IBDs.