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.

P2X7 receptor signaling during adult hippocampal neurogenesis

Neurogenesis is a persistent and essential feature of the adult mammalian hippocampus.Granular neurons generated from resident pools of stem or progenitor cells provide a mechanism for the formation and consolidation of new memories.Regulation of hippocampal neurogenesis is complex and multifaceted,and numerous signaling pathways converge to modulate cell proliferation,apoptosis,and clearance of cellular debris,as well as synaptic integration of newborn immature neurons.The expression of functional P2X7 receptors in the central nervous system has attracted much interest and the regulatory role of this purinergic receptor during adult neurogenesis has only recently begun to be explored.P2X7 receptors are exceptionally versatile:in their canonical role they act as adenosine triphosphate-gated calcium channels and facilitate calcium-signaling cascades exerting control over the cell via calcium-encoded sensory proteins and transcription factor activation.P2X7 also mediates transmembrane pore formation to regulate cytokine release and facilitate extracellular communication,and when persistently stimulated by high extracellular adenosine triphosphate levels large P2X7 pores form,which induce apoptotic cell death through cytosolic ion dysregulation.Lastly,as a scavenger receptor P2X7 directly facilitates phagocytosis of the cellular debris that arises during neurogenesis,as well as during some disease states.Understanding how P2X7 receptors regulate the physiology of stem and progenitor cells in the adult hippocampus is an important step towards developing useful therapeutic models for regenerative medicine.This review considers the relevant aspects of adult hippocampal neurogenesis and explores how P2X7 receptor activity may influence the molecular physiology of the hippocampus,and neural stem and progenitor cells.

Enteric nervous system and inflammatory bowel diseases:Correlated impacts and therapeutic approaches through the P2X7 receptor

The enteric nervous system(ENS)consists of thousands of small ganglia arranged in the submucosal and myenteric plexuses,which can be negatively affected by Crohn’s disease and ulcerative colitis-inflammatory bowel diseases(IBDs).IBDs are complex and multifactorial disorders characterized by chronic and recurrent inflammation of the intestine,and the symptoms of IBDs may include abdominal pain,diarrhea,rectal bleeding,and weight loss.The P2X7 receptor has become a promising therapeutic target for IBDs,especially owing to its wide expression and,in the case of other purinergic receptors,in both human and model animal enteric cells.However,little is known about the actual involvement between the activation of the P2X7 receptor and the cascade of subsequent events and how all these activities associated with chemical signals interfere with the functionality of the affected or treated intestine.In this review,an integrated view is provided,correlating the structural organization of the ENS and the effects of IBDs,focusing on cellular constituents and how therapeutic approaches through the P2X7 receptor can assist in both protection from damage and tissue preservation.

P2X7 receptor as the regulator of T-cell function in intestinal barrier disruption

The intestinal mucosa is a highly compartmentalized structure that forms a directbarrier between the host intestine and the environment, and its dysfunction couldresult in a serious disease. As T cells, which are important components of themucosal immune system, interact with gut microbiota and maintain intestinalhomeostasis, they may be involved in the process of intestinal barrier dysfunction.P2X7 receptor (P2X7R), a member of the P2X receptors family, mediates the effectsof extracellular adenosine triphosphate and is expressed by most innate or adaptiveimmune cells, including T cells. Current evidence has demonstrated thatP2X7R is involved in inflammation and mediates the survival and differentiationof T lymphocytes, indicating its potential role in the regulation of T cell function.In this review, we summarize the available research about the regulatory role andmechanism of P2X7R on the intestinal mucosa-derived T cells in the setting ofintestinal barrier dysfunction.

P2X7 receptor antagonist recovers ileum myenteric neurons after experimental ulcerative colitis

BACKGROUND The P2X7 receptor is expressed by enteric neurons and enteric glial cells.Studies have demonstrated that administration of a P2X7 receptor antagonist,brilliant blue G(BBG),prevents neuronal loss.AIM To report the effects of BBG in ileum enteric neurons immunoreactive(ir)following experimental ulcerative colitis in Rattus norvegicus albinus.METHODS 2,4,6-trinitrobenzene sulfonic acid(TNBS group,n=5)was injected into the distal colon.BBG(50 mg/kg,BBG group,n=5)or vehicle(sham group,n=5)was given subcutaneously 1 h after TNBS.The animals were euthanized after 24 h,and the ileum was removed.Immunohistochemistry was performed on the myenteric plexus to evaluate immunoreactivity for P2X7 receptor,neuronal nitric oxide synthase(nNOS),choline acetyltransferase(ChAT),HuC/D and glial fibrillary acidic protein.RESULTS The numbers of nNOS-,ChAT-,HuC/D-ir neurons and glial fibrillary acidic protein-ir glial cells were decreased in the TNBS group and recovered in the BBG group.The neuronal profile area(μm^2)demonstrated that nNOS-ir neurons decreased in the TNBS group and recovered in the BBG group.There were no differences in the profile areas of ChAT-and HuC/D-ir neurons.CONCLUSION Our data conclude that ileum myenteric neurons and glial cells were affected by ulcerative colitis and that treatment with BBG had a neuroprotective effect.Thus,these results demonstrate that the P2X7 receptor may be an important target in therapeutic strategies.

P2X7 receptor inhibitor suppressed extracellular ATP/LPS-primed human hepatic stellate cells activation via downregulating NLRP3 inflammasome

OBJECTIVE To investigate the effect of P2X7receptor(P2X7r)inhibition,using a specific inhibitor(A438079)to prevent the development of liver fibrosis on human hepatic stellate cells,LX-2.METHODS The supernatant from lipopolysaccharide(LPS)-stimulated RAW264.7 mouse macrophages was supplemented to LX-2 cells for 24 h.LX-2cells were primed with LPS for 4h and subsequently stimulated for 30 min with 3mmol·L-1 of adenosine 5′-triphosphate(ATP).A438079(10μmol·L-1)was supplemented to LX-2 cells 10 min prior to ATP.RESULTS Directly treated with LPS on LX-2 cells,mRNA expressions of IL-1β,IL-18 and IL-6 were increased,as well as P2X7 r.And caspase-1,ASC and NLRP3 mRNA expressions were increased with LPS stimulation.LPS stimulation also increasedα-SMA and collagenⅠ mRNA expressions.Interestingly treatment of LX-2cells with mediums from LPS-primed RAW264.7mouse macrophages exhibited greater increase of mRNA expressions of above genes than those in LX-2directly treated with LPS.Pretreatment of directly or indirectly LPS-stimulated LX-2 cells with A438079 both suppressed IL-1βmRNA expression.In addition treatment of LPS-primed LX-2 cells with 3mmol·L-1 ATP induced the significant increase of IL-1β,IL-6,caspase-1,pannexin-1,α-SMA and collagenⅠ mRNA expression,the increasing ofα-SMA protein expression and cleavage of IL-1β.These events were significantly suppressed by pretreatment with P2X7 rantagonist A438079.P2X7 rblockade also significantly reduced the protein expression ofα-SMA.CONCLUSION Our results suggest that the involvement of the P2X7r-NLRP3 inflammasome pathway in the secretion of IL-1βfrom extracellular ATP/LPS-stimulated human hepatic stellate cells.This study demonstrated that repression of the P2X7 rrepresents a novel potential therapeutic approach to control liver fibrosis.

P2X7 receptor blockade decreases inflammation,apoptosis,and enteric neuron loss during Clostridioides difficile toxin A-induced ileitis in mice

Clostridioides difficile(C.difficile)is the most common pathogen causing health care-associated infections.C.difficile TcdA and TcdB have been shown to activate enteric neurons;however,what population of these cells is more profoundly influenced and the mechanism underlying these effects remain unknown.AIM To characterize a specific population of TcdA-affected myenteric neurons and investigate the role of the P2X7 receptor in TcdA-induced ileal inflammation,cell death,and the changes in the enteric nervous system in mice.METHODS Swiss mice were used to model TcdA-induced ileitis in ileal loops exposed to TcdA(50μg/Loop)for 4 h.To investigate the role of the P2X7 receptor,Brilliant Blue G(50 mg/kg,i.p.),which is a nonspecific P2X7 receptor antagonist,or A438079(0.7μg/mouse,i.p.),which is a competitive P2X7 receptor antagonist,were injected one hour prior to TcdA challenge.Ileal samples were collected to analyze the expression of the P2X7 receptor(by quantitative real-time polymerase chain reaction and immunohistochemistry),the population of myenteric enteric neurons(immunofluorescence),histological damage,intestinal inflammation,cell death(terminal deoxynucleotidyltransferasemediated dUTP-biotin nick end labeling),neuronal loss,and S100B synthesis(immunohistochemistry).RESULTS TcdA upregulated(P<0.05)the expression of the P2X7 receptor gene in the ileal tissues,increasing the level of this receptor in myenteric neurons compared to that in control mice.Comparison with the control mice indicated that TcdA promoted(P<0.05)the loss of myenteric calretinin+(Calr)and choline acetyltransferase+neurons and increased the number of nitrergic+and Calr+neurons expressing the P2X7 receptor.Blockade of the P2X7 receptor decreased TcdAinduced intestinal damage,cytokine release[interleukin(IL)-1β,IL-6,IL-8,and tumor necrosis factor-α],cell death,enteric neuron loss,and S100B synthesis in the mouse ileum.CONCLUSION Our findings demonstrated that TcdA induced the upregulation of the P2X7 receptor,which promoted enteric neuron loss,S100B synthesis,tissue damage,inflammation,and cell death in the mouse ileum.These findings contribute to the future directions in understanding the mechanism involved in intestinal dysfunction reported in patients after C.difficile infection.

Targeting the P2X7 receptor in microglial cells to prevent brain inflammation

Microglial cells are the key innate immune cells in the brain and they are crucial in maintaining brain parenchyma homeostasis.Under physiological conditions,microglial cells assume a ramified morphology with a small cell body and an extensive network of fine processes,which secrete neurotrophic factors and patrol the surroundings in search for pathogens and eliminate cellular debris via phagocytosis.Microglial cells express a repertoire of pattern recognition receptors(PRRs)that enable them to detect diverse danger-associated molecular patterns(DAMPs)released from damaged cells or cells under stress,or pathogen-associated molecular patterns generated by pathogens during infection.

P2X7 receptor activation causes phosphatidylserine exposure in canine erythrocytes

AIM To determine if activation of the ATP-gated P2X7 receptor channel induces phosphatidylserine(PS) exposure in erythrocytes from multiple dog breeds.METHODS Peripheral blood was collected from 25 dogs representing 13 pedigrees and seven crossbreeds. ATP-induced PS exposure on canine erythrocytes in vitro was assessed using a flow cytometric Annexin V binding assay.RESULTS ATP induced PS exposure in erythrocytes from all dogs studied. ATP caused PS exposure in a concentrationdependent manner with an EC50 value of 395 μmol/L. The non-P2X7 agonists, ADP or AMP, did not cause PS exposure. The P2X7 antagonist, AZ10606120, but not the P2X1 antagonist, NF449, blocked ATP-induced PS exposure.CONCLUSION The results indicate that ATP induces PS exposure in erythrocytes from various dog breeds and that this process is mediated by P2X7 activation.

P2X7 Receptor Mediated Growth-Inhibitory Effect in KG1a Cell Line

OBJECTIVE This study was conducted to investigate ATP- induced growth inhibition in human leukemic cells KG1a. METHODS ATP inhibited cell growth was analyzed by MTS assay. Externalization of phosphatidylserine could be detected by Annexin-V-FITC apoptosis staining after activation of the P2X7 receptor. P2X7 mediated pore formation was detected in KGla cells by Yo-Pro-1 uptake assay. RESULTS ATP inhibited cell growth in a dose-dependent manner. The cytotoxic effect could be blocked by P2X7 antagonists, oxidized ATP （oATP） and KN62. Externalization of phosphatidylserine could be detected in a time-dependent manner. P2X7 mediated pore formation could be detected in KG1a cells. These effects could not be observed in P2X7 null Ramos cells. CONCLUSION The results and our previously reports that mRNA, protein expression and calcium response of the P2X7 receptor in KGla cells, suggested that extracellular ATP effectively induces growth inhibition through apoptosis in KGla cells by activation of P2X7 receptor, and that may be mediated by extracellular Ca^2＋ in ux and pore formation.

P2X7 receptor in skin biology and diseases

The P2X7 receptor is a trimeric ligand-gated cation channel present on immune and other cells. Activation of this receptor by its natural ligand extracellular adenosine triphosphate results in a variety of downstream responses, including the release of pro-inflammatory mediators and cell death. In normal skin, P2X7 is present on keratinocytes, Langerhans cells and fibroblasts, while the presence of this receptor on other cutaneous cells is mainly inferred from studies of equivalent cell types present in other tissues. Mast cells in normal skin however express negligible amounts of P2X7, which can be upregulated in cutaneous disease. This review discusses the potential significance of P2X7 in skin biology, and the role of this receptor in inflammatory skin disorders such as irritant and chronic dermatitis, psoriasis, graft-versus-host disease, as well is in wound healing, transplantation and skin cancer.

Correlation of serum P2X7 receptor, CD64 and CD54 expression with infection process in children with bacterial pneumonia

Objective:To investigate the correlation of serum P2X7 receptor, CD64 and CD54 expression with infection process in children with bacterial pneumonia.Methods: A total of 164 children with bacterial pneumonia hospitalized in this hospital between June 2016 and February 2018 were selected as bacterial pneumonia group, and 100 healthy children who received vaccination in this hospital during the same period were selected as normal control group. The expression levels of P2X7 receptor, CD64 and CD54 as well as the contents of inflammatory factors, acute phase proteins and immunoglobulins in serum of the two groups were detected. Results: Immediately after admission, serum P2X7 receptor, CD64 and CD54 expression of bacterial pneumonia group were higher than those of normal control group, inflammatory cytokines TNF-α, sTREM-1, IL-2 and IL-6 contents were higher than those of normal control group, acute phase proteins 1-AGP, CRP, CP and HP contents were higher than those of normal control group, and immunoglobulins IgA, IgM and IgG contents were higher than those of normal control group;serum P2X7 receptor, CD64 and CD54 expression in children with bacterial pneumonia were positively correlated with TNF-α, sTREM-1, IL-2, IL-6, 1-AGP, CRP, CP, HP, IgA, IgM and IgG contents.Conclusion:The serum P2X7 receptor, CD64 and CD54 expression increase in children with bacterial pneumonia, and they are positively correlated with the degree of infection.

P2X7受体抑制剂腹腔注射对红藻氨酸诱导小鼠癫痫发作及海马损伤的影响

目的观察P2X7受体(P2X7R)抑制剂对红藻氨酸诱导小鼠癫痫发作及海马损伤的影响。方法雄性C57BL/6小鼠96只,随机分为正常组36只、模型组36只、P2X7R抑制剂组24只。P2X7R抑制剂组于造模前30 min腹腔注射P2X7R特异性抑制剂A438079,模型组、正常组在同时点注射等量生理盐水。模型组、P2X7R抑制剂组腹腔注射红藻氨酸建立癫痫模型,正常组腹腔注射等量生理盐水。采用脑电图观察小鼠癫痫发作情况,Western blotting法检测小鼠海马组织P2X7R蛋白,尼氏染色观察小鼠海马CA1区损伤情况。采用旷场实验观察正常组、模型组小鼠焦虑样行为,若模型组存在焦虑样行为,则进一步将模型组分为焦虑对照亚组及P2X7R抑制亚组,P2X7R抑制亚组每天腹腔注射A438079,焦虑对照亚组每天腹腔注射等量生理盐水,持续14 d后再次进行旷场实验。采用水迷宫实验观察小鼠认知功能,若模型组存在认知功能损害,则进一步将模型组分为认知功能对照亚组及P2X7R抑制亚组,处理方式同上,持续14 d后再次进行水迷宫实验。结果脑电图记录结果显示,正常组未出现癫痫脑电图波形;模型组出现癫痫持续状态,脑电图表现出强放电功率及高振幅;P2X7R抑制剂组出现癫痫发作,但脑电图放电功率及振幅均较模型组减轻;脑电图总功率及平均振幅模型组>P2X7R抑制剂组>正常组(P均<0.05)。模型组海马组织P2X7R蛋白表达高于正常组,P2X7R抑制剂组海马组织P2X7R蛋白表达低于模型组(P均<0.05)。尼氏染色结果显示,对照组海马CA1区结构完整,存活神经细胞数多,尼氏体较大且较多;模型组海马CA1区结构完整性被破坏,神经细胞数量少,尼氏体数量减少;P2X7R抑制剂组海马CA1区结构、神经细胞及尼氏体数量均较模型组有所改善。旷场实验结果显示,模型组进入中心区域次数及中心区域停留时间均少于正常组,焦虑对照亚组进入中心区域次数及中心区域停留时间均少于P2X7R抑制亚组(P均<0.05)。水迷宫实验结果显示,模型组、正常组穿越平台次数及目标象限停留时间比较差异均无统计学意义(P均>0.05)。结论P2X7R抑制剂可减轻红藻氨酸诱导小鼠癫痫发作的强度和频率,改善癫痫所致海马损伤,且可缓解癫痫小鼠的焦虑样行为。

P2X7受体在癌痛中作用的研究进展

P2X7受体是一种ATP门控离子通道,在肿瘤条件下,P2X7受体的激活触发了促炎细胞因子的释放,刺激了伤害性神经元的兴奋,从而加剧了疼痛的传递。在临床前癌疼痛模型中,具有作为癌疼痛管理的新治疗靶点的潜力。

阻塞性睡眠呼吸暂停患者血清P2X7R、NF-κB水平与认知功能障碍的相关性

目的 研究阻塞性睡眠呼吸暂停(OSA)患者血清P2X7受体(P2X7R)和核因子-κB(NF-κB)水平与认知功能障碍的相关性。方法 选择2021年10月至2022年12月期间青海省人民医院收治的126例OSA患者作为OSA组,进一步根据蒙特利尔认知评估量表(MoCA)评分分为MoCA评分<26分的认知功能障碍组(n=68)和MoCA评分≥26分的认知功能正常组(n=58);另取同期体检的健康志愿者作为对照组(n=105)。检测血清P2X7R、NF-κB水平,采用logistic回归模型分析OSA患者认知功能障碍的影响因素,采用ROC曲线分析各指标对OSA患者认知功能障碍的诊断价值。结果 OSA组患者的血清P2X7R、NF-κB水平高于对照组,差异有统计学意义(t=17.295、7.088,P<0.05);认知功能障碍组患者的血清P2X7R、NF-κB水平高于认知功能正常组,差异有统计学意义(t=8.469、8.497,P<0.05);血清P2X7R、NF-κB水平升高是OSA患者认知功能障碍的危险因素(P<0.05);OSA患者血清P2X7R、NF-κB水平与MoCA评分呈负相关(P<0.05);血清P2X7R、NF-κB水平诊断OSA患者认知功能障碍的曲线下面积分别为0.862(95%CI:0.800~0.923,P<0.05)和0.859(95%CI:0.794~0.923)。结论 OSA患者血清P2X7R、NF-κB水平升高且与认知功能障碍相关,血清P2X7R、NF-κB对OSA患者认知功能障碍具有诊断效能。

亮蓝G通过抑制P2X7/NLRP3通路减轻脑缺血再灌注大鼠神经炎症

目的:研究嘌呤受体P2X7在脑缺血再灌注损伤(CIRI)中的作用。方法:使用右侧大脑中动脉闭塞法(MCAO)制备大鼠CIRI模型,通过腹腔注射给予P2X7抑制剂亮蓝G(BBG)或NLRP3抑制剂MCC950处理。神经功能评分检测大鼠神经功能的改变,伊文思蓝染色检测血脑屏障完整性,通过酶联免疫吸附试验(ELISA)检测大鼠脑脊液中IL-1β和IL-18的含量,通过Western Blot检测右侧大脑皮质中P2X7、CD11b和NLRP3的表达。结果:BBG或者MCC950处理能改善CIRI大鼠神经功能,同时降低脑组织中伊文思蓝的含量,并降低脑脊液中IL-1β和IL-18的水平。此外,BBG可以降低右侧大脑皮质中CD11b和NLRP3的表达,但是MCC950只能降低CD11b表达,对P2X7表达没有明显影响。结论:BBG通过抑制P2X7/NLRP3通路减轻CIRI大鼠神经炎症。

P2X7受体在心血管疾病中的研究进展

P2X7受体,作为嘌呤受体P2X家族的一员,主要在免疫细胞、心肌平滑肌细胞和内皮细胞中表达。其天然配体ATP在组织缺氧或炎症状态下释放量显著增加。P2X7受体与ATP结合后可激活核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)依赖炎症小体,从而加剧炎症反应。近期研究揭示了P2X7受体在动脉粥样硬化、高血压、心肌梗死和肺动脉高压等心血管疾病中的病理作用。本文旨在综述P2X7受体的分子结构、分布、功能特性,及其在心血管疾病中的研究进展。

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。

冬凌草甲素对海马CA1星形胶质细胞P2X7受体电流的影响

目的:探索冬凌草甲素是否对海马CA1星形胶质细胞P2X7受体电流有影响。方法:采用Autodock软件对冬凌草甲素与P2X7蛋白进行分子对接;采用全细胞膜片钳记录冬凌草甲素对海马CA1星形胶质细胞P2X7受体激动剂Bz-ATP激发电流的影响。结果:冬凌草甲素与P2X7蛋白的结合能为-5.86 kcal/mol,可与P2X7蛋白的Lys592残基形成氢键,与Tyr550残基形成疏水作用;Bz-ATP能激发海马CA1区星形胶质细胞的电流反应,且1000μmol/L Bz-ATP激发的电流反应较强;10μmol/L冬凌草甲素溶液孵育1 h后,海马CA1区星形胶质细胞对谷氨酸能受体激动剂NMDA和AMPA溶液的电流反应无明显变化;对Bz-ATP的电流反应降低,差异具有统计学意义(P<0.05)。结论:冬凌草甲素可与P2X7蛋白对接形成稳定的复合物,具有较好的亲和力;冬凌草甲素可抑制海马CA1星形胶质细胞P2X7受体电流。

细胞外ATP激活受体P2X7在骨质疏松症中的作用机制

骨质疏松症已逐渐成为一个重大的医疗和社会经济问题,其典型特征包括骨密度下降、骨微结构损坏以及骨折风险的增加。现有的治疗策略主要集中在症状管理上,但其并未能从根本上改变病情的发展趋势。因此,寻找并开发针对新的治疗靶点是更有效的治疗策略,已经成为当前研究的重要方向。P2X7作为一种由胞外ATP激活的离子通道,在近年的研究中已证实其在骨形成细胞和骨吸收细胞的生长、功能和存活方面发挥了关键作用,对于骨质疏松症的治疗具有巨大的潜力。因此,P2X7有望成为骨质疏松症治疗的新靶点。笔者对P2X7在骨质疏松症中的作用以及如何针对这一新靶点的治疗进行综述,旨在为开发新的骨质疏松症治疗策略提供新的思路和方向。