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.

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 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 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 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 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 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.

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。

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。

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。

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。

P2X7受体在肿瘤发生发展中的作用研究进展

P2X7受体(P2X7 Purinergic Receptor,P2X7R)是一种非选择性三聚体阳离子通道,受胞外ATP激活而开放。其具有对三磷酸腺苷(Adenosine Triphosphate,ATP)亲和力较低、不易脱敏及大孔开放等特征。P2X7R广泛表达于肿瘤细胞和免疫细胞中,被认为与肿瘤密切相关,是潜在的抗肿瘤药物靶点。多数观点认为P2X7R在肿瘤中的表达上调,其抑制剂可以抑制肿瘤发生发展,但由于P2X7R对肿瘤微环境(Tumor Microenvironment,TME)尤其是TME中的免疫细胞会产生复杂影响,导致P2X7R抑制剂临床研究进展缓慢。本文主要从P2X7R对肿瘤以及TME产生的影响入手,总结了近年来的研究进展。

利多卡因对三叉神经痛大鼠p2x7-p38-IL-Iβ通路的影响

目的探讨利多卡因对三叉神经痛p2x7-p38-IL-Iβ通路的影响。方法雄性SD大鼠随机数字表法分为3组:假手术组、手术组(ION-CCI组)、利多卡因组(ION-CCI+L组)。假手术组:钝性分离右侧眶下神经但不结扎+腹腔注射等体积0.9%氯化钠注射液;ION-CCI组:分离右侧眶下神经并结扎+腹腔注射等体积0.9%氯化钠注射液;ION-CCI+L组:分离右侧眶下神经并结扎+腹腔注射利多卡因10 mg·kg-1·d-1。术前、术后1 d、3 d、5 d、7 d、9 d、11 d、14 d行大鼠机械痛阈测定。术后14 d腹腔注射戊巴比妥钠(60 mg/kg)麻醉并灌注取脑组织、三叉神经节。采用酶联免疫吸附试验(ELISA)检测大鼠丘脑、三叉神经节白细胞介素(IL)-1β的表达水平,蛋白质免疫印迹法检测大鼠丘脑P2X7、p38、p-p38、IL-1β的表达水平,免疫荧光检测大鼠丘脑p-p38的表达水平。结果ION-CCI+L组大鼠痛阈较假手术组降低(P<0.01),较ION-CCI组增高(P<0.01);ELISA结果显示,ION-CCI+L组大鼠丘脑、三叉神经节IL-1β浓度较ION-CCI组降低(P<0.05),较假手术组增高(P<0.05);蛋白质免疫印迹法结果显示ION-CCI+L组大鼠丘脑p2x7、p-p38的表达水平较ION-CCI组降低(P<0.01)、较假手术组增高(P<0.01),ION-CCI+L组大鼠丘脑IL-1β的表达水平较ION-CCI组降低(P<0.05)、较假手术组增高(P<0.01)、各组大鼠丘脑p38的表达水平差异无统计学意义(P>0.05);免疫荧光结果显示ION-CCI+L组大鼠丘脑区p-p38的荧光强度较ION-CCI组降低(P<0.05)、较假手术组增高(P<0.05)。结论利多卡因可缓解大鼠三叉神经痛,降低三叉神经痛大鼠丘脑和三叉神经节IL-1β的表达水平、降低丘脑p2x7、p-p38的表达水平,提示利多卡因可能通过抑制P2X7-p38-IL-1β通路抑制中枢神经系统炎症反应从而缓解三叉神经痛。

Sleep Deprivation Selectively Down-Regulates Astrocytic 5-HT2B Receptors and Triggers Depressive-Like Behaviors via Stimulating P2X7 Receptors in Mice

Chronic loss of sleep damages health and disturbs the quality of life.Long-lasting sleep deprivation(SD)as well as sleep abnormalities are substantial risk factors for major depressive disorder,although the underlying mechanisms are not clear.Here,we showed that chronic SD in mice promotes a gradual elevation of extracellular ATP,which activates astroglial P2X7 receptors(P2X7Rs).Activated P2X7Rs,in turn,selectively down-regulated the expression of 5-HT2B receptors(5-HT2BRs)in astrocytes.Stimulation of P2X7Rs induced by SD selectively suppressed the phosphorylation of AKT and FoxO3 a in astrocytes,but not in neurons.The overexpression of FoxO3a in astrocytes inhibited the expression of 5-HT2BRs.Down-regulation of 5-HT2BsRs instigated by SD suppressed the activation of STAT3 and relieved the inhibition of Ca2+-dependent phospholipase A2.This latter cascade promoted the release of arachidonic acid and prostaglandin E2.The depression-like behaviors induced by SD were alleviated in P2X7R-KO mice.Our study reveals the mechanism underlying chronic SD-induced depression-like behaviors and suggests 5-HT2BRs as a key target for exploring therapeutic strategies aimed at the depression evoked by sleep disorders.

P2X7 Receptor Antagonism Attenuates the Intermittent Hypoxia-induced Spatial Deficits in a Murine Model of Sleep Apnea Via Inhibiting Neuroinflammation and Oxidative Stress

Background：The mechanism of the neural injury caused by chronic intermittent hypoxia （CIH） that characterizes obstructive sleep apnea syndrome （OSAS） is not clearly known.The purpose of this study was to investigate whether P2X7 receptor （P2X7R） is responsible for the CIH-induced neural injury and the possible pathway it involves.Methods：Eight-week-old male C57BL/6 mice were used.For each exposure time point,eight mice divided in room air （RA） and IH group were assigned to the study of P2X7R expression.Whereas in the 21 days-Brilliant Blue G （BBG,a selective P2X7R antagonist） study,48 mice were randomly divided into CIH group,BBG-treated CIH group,RA group and BBG-treated RA group.The hippocampus P2X7R expression was determined by Western blotting and real-time polymerase chain reaction （PCR）.The spatial learning was analyzed by Morris water maze.The nuclear factor kappa B （NFκB） and NADPH oxidase 2 （NOX2） expressions were analyzed by Westem blotting.The expressions of tumor necrosis factor α,interleukin 1 β （IL-β）,IL-18,and IL-6 were measured by real-time PCR.The malondialdehyde and superoxide dismutase levels were detected by colorimetric method.Cell damage was evaluated by Hematoxylin and Eosin staining and Terminal Transferase dUTP Nick-end Labeling method.Results：The P2X7R mRNA was elevated and sustained after 3-day IH exposure and the P2X7R protein was elevated and sustained after 7-day IH exposure.In the BBG study,the CIH mice showed severer neuronal cell damage and poorer performance in the behavior test.The increased NFκB and NOX2 expressions along with the inflammation injury and oxidative stress were also observed in the CIH group.BBG alleviated CIH-induced neural injury and consequent functional deficits.Conclusions：The P2X7R antagonism attenuates the CIH-induced neuroinflammation,oxidative stress,and spatial deficits,demonstrating that the P2X7R is an important therapeutic target in the cognition deficits accompanied OSAS.

Paraventricular Nucleus P2X7 Receptors Aggravate Acute Myocardial Infarction Injury via ROS-Induced Vasopressin-V1b Activation in Rats

The present study was designed to investigate the mechanisms by which P2X7 receptors(P2X7Rs)mediate the activation of vasopressinergic neurons thereby increasing sympathetic hyperactivity in the paraventricular nucleus(PVN) of the hypothalamus of rats with acute myocardial ischemia(AMI). The left anterior descending branch of the coronary artery was ligated to induce AMI in rats. The rats were pretreated with BBG(brilliant blue G, a P2X7R antagonist), nelivaptan(a vasopressin V1b receptor antagonist), or diphenyleneiodonium(DPI) [an nicotinamide adenine dinucleotide phosphate(NADPH)oxidase inhibitor]. Hemodynamic parameters of the heart were monitored. Myocardial injury and cardiomyocyte apoptosis were assessed. In the PVN of AMI rats, P2X7R mediated microglial activation, while reactive oxygen species(ROS) and NADPH oxidase 2(NOX2) were higher than in the sham group. Intraperitoneal injection of BBG effectively reduced ROS production and vasopressin expression in the PVN of AMI rats. Moreover, both BBG and DPI pretreatment effectively reduced sympathetic hyperactivity and ameliorated AMI injury, as represented by reduced inflammation and apoptosis of cardiomyocytes.Furthermore, microinjection of nelivaptan into the PVN improved cardiac function and reduced the norepinephrine(AE) levels in AMI rats. Collectively, the results suggest that, within the PVN of AMI rats, P2X7R upregulation mediates microglial activation and the overproduction of ROS, which in turn activates vasopressinergic neuron V1b receptors and sympathetic hyperactivity, hence aggravating myocardial injury in the AMI setting.

P2X7 Receptor in Alcoholic Steatohepatitis and Alcoholic Liver Fibrosis

Alcoholic liver disease is one of the most common chronic liver diseases in the world.It is a liver disease caused by prolonged heavy drinking and its main clinical features are nausea,vomiting,enlargement of the liver,and jaundice.Recent studies suggest that Kupffer cell-mediated inflam-matory response is a core driver in the development of alco-holic steatohepatitis and alcoholic liver fibrosis.As a danger signal,extracellular ATP activates the assembly of NLPR3 inflammasome by acting on purine P2X7 receptor,the ac-tivated NLRP3 inflammasome prompts ASC to cleave pro-cCaspase-1 into active caspase-1in KCs.Active caspase-1 promotes the conversion of pro-IL-1βto IL-1β,which fur-ther enhances the inflammatory response.Here,we briefly review the role of the P2X7R-NLRP3 inflammasome axis in the pathogenesis of alcoholic liver disease and the evolution of alcoholic steatohepatitis and alcoholic liver fibrosis.Reg-ulation of the inflammasome axis of P2X7R-NLRP3 may be a new approach for the treatment of alcoholic liver disease.