组胺H3受体参与低氧调控的研究进展

低氧是指机体供氧不足或用氧障碍,栓死、一氧化碳中毒和阻塞性呼吸睡眠等多种病理过程均与低氧相关。高原地区特有的低氧环境会使人因供氧不足而产生急性高原反应,甚至高原肺水肿和脑水肿。组胺H3受体广泛分布于中枢及外周组织,主要作为突触前膜受体,调控组胺及多巴胺、乙酰胆碱等多种神经递质的释放。低氧条件下组胺H3受体可分别于颈动脉体及呼吸中枢延髓参与呼吸活动的调控。此外,组胺能系统在中枢参与低血低氧性脑损伤调控,其中组胺H3受体发挥了重要作用。本文就组胺H3受体的结构特征、功能及其参与低氧调控的可能机制做一综述,阐述靶向组胺H3受体研发抗低氧药物的可能性。     

组胺H2受体激动剂和拮抗剂及抗癌药对正常人造血祖细胞和HL-60白血病细胞生长的作用

采用体外微量克隆培养体系研究了组胺H2受体激动剂4-甲基组胺(4-MH)和拮抗剂雷尼替叮(ranitidine)及抗癌药阿糖胞苷分别对正常人外周血粒-巨噬系祖细胞(PBCFU-GM)和HL-60白血病细胞生长的作用。当4-MH的浓度为10^-9～10^-6mol·L^-1时,可促进PBCFU-GM的增殖,4-MH的浓度增加至10^-4mol·L^-1时则表现为抑制PBCFU-GM的增殖。Ranitidine的浓度为10^-9～10^-5mol·L^-1时,表现出对PBCFU-GM增殖的抑制作用,但在10-6mol·L^-1剂量时对PBCFU-GM的抑制率低于50%,而在该剂量时对HL-60白血病细胞的抑制率已达100%,具有一定的选择性。抗癌药阿糖胞苷(Ara-C)对HL-60白血病细胞的抑制作用比对PBCFU-GM的抑制作用较强,但两者的IC₅₀值处于同一个数量级。在强化化疗剂量10^-5mol·L^-1时,Ara-C对HL-60白血病细胞和PBCFU-GM正常造血祖细胞的抑制率均达100%。     

五步蛇毒对大鼠海马组胺受体H_1 mRNA和蛋白质表达的影响

目的研究五步蛇毒对大鼠海马组胺受体H1mRNA及蛋白质表达的影响。方法用半定量RT-PCR和Western blot方法,检测腹腔注射五步蛇毒后不同时相大鼠海马组胺受体H1mRNA及蛋白质表达的变化。结果五步蛇毒处理后,大鼠海马组胺受体H1mRNA及蛋白质的表达在各时相点均发生变化,且其mRNA和蛋白质表达均为先下调后上调。结论五步蛇毒对大鼠脑区海马组胺H1受体mRNA和蛋白质表达都有影响。H1受体mRNA和蛋白质表达的改变对大脑的生理功能有一定的影响,可能是该蛇毒致脑功能障碍的分子基础之一。     

M3受体对体外H2O2诱导大鼠心肌细胞凋亡的保护作用

目的探讨M₃受体激动对H₂O₂诱导的大鼠培养心肌细胞凋亡的作用，进一步阐明其机制。方法末端标记法 (TUNEL)进行细胞凋亡检测；免疫组化方法检测Bcl-2和Fas的表达；共聚焦显微镜观察［Ca²⁺］_i荧光强度变化。结果M₃受体激动剂胆碱(10 mmol·L^-1)可减少H₂O₂诱导的心肌细胞凋亡的数量，并可增加心肌Bcl-2的表达，减少Fas表达，抑制H₂O₂诱导的［Ca²⁺］_i荧光强度的升高。但预先应用4DAMP (10 nmol·L^-1)阻断M₃受体可逆转胆碱作用。结论激动M₃受体对H₂O₂诱导的心肌细胞凋亡有保护作用，其机制可能与Bcl-2和Fas表达以及下调［Ca²⁺］_i有关。     

钙蛋白酶抑制剂calpeptin对大鼠海马脑片缺氧无糖损伤的保护作用

目的通过观察电生理学和神经元凋亡的变化,研究钙蛋白酶抑制剂calpeptin对大鼠离体海马脑片缺氧无糖损伤的保护作用。方法40片SD大鼠海马脑片随机分为两组:对照组和calpeptin组。根据人工脑脊液中calpeptin的浓度,再细分为1μmol.L-1组、10μmol.L-1组、100μmol.L-1组和200μmol.L-1组,每组8片脑片。采用细胞外记录技术观察calpeptin对大鼠离体海马脑片在缺氧无糖条件下顺向群峰电位(orthodromicpopulationspikes,OPS)及缺氧损伤电位(hypoxicinjurypotential,HIP)变化的影响,采用TUNEL法观察缺氧无糖损伤后海马脑片CA1区锥体细胞凋亡情况及calpeptin对它的影响。结果10μmol.L-1组、100μmol.L-1组和200μmol.L-1组HIP出现率及海马CA1区锥体细胞层凋亡细胞数减少,OPS恢复率和OPS恢复程度与对照组比较提高。而10μmol.L-1组、100μmol.L-1组和200μmol.L-1组之间各项指标差异无显著性。结论(10～200)μmol.L-1calpeptin可以减轻大鼠海马脑片的缺氧无糖损伤,其机制可能与calpeptin减少海马CA1区神经元凋亡有关。     

ERK1/2的激活参与七氟醚预处理对大鼠海马脑片缺氧无糖损伤的保护作用

目的观察ERK1/2的激活在七氟醚预处理对大鼠海马脑片缺氧无糖损伤保护中的作用。方法采用脑片灌流及电生理技术,细胞外记录海马CA1区的顺向群锋电位(OPS);利用2,3,5-三苯基氯化四氮唑(TTC)染色定量比色方法分析脑片损伤程度。结果用4%七氟醚预处理海马脑片,可延迟OPS的消失时间,提高复氧后OPS的恢复程度和恢复率。以ERK1/2特异性抑制剂PD98059(50μmol.L-1)预处理海马脑片,可以取消七氟醚的作用。单独使用PD98059对OPS无明显影响。七氟醚预处理组组织损伤百分率明显低于其它各组。结论ERK1/2的激活参与了七氟醚预处理对大鼠海马脑片缺氧无糖损伤的保护作用。     

依达拉奉、米诺环素及ONO-1078对缺氧缺糖诱导大鼠海马脑片电生理改变的作用

目的：建立离体海马脑片缺氧缺糖(OGD)电生理变化模型，观察依达拉奉、米诺环素和ONO-1078{pranlukast，4-氧-8-[对-(4-苯丁氧基)苯甲酰氨基]-2-(5-四氮基)-4H-1-苯并吡喃半水化合物}的神经保护作用。方法：大鼠海马脑片以无氧无糖处理，记录脑片群峰电位(PS)，部分实验以TIC染色     

依达拉奉、米诺环素及ONO-1078对缺氧缺糖诱导大鼠海马脑片电生理改变的作用

目的建立离体海马脑片缺氧缺糖(OGD)电生理变化模型,观察依达拉奉、米诺环素和ONO-1078 {pranlukast,4-氧-8-［对-(4-苯丁氧基)苯甲酰氨基］-2-(5-四氮基)-4H-1-苯并吡喃半水化合物}的神经保护作用。方法 大鼠海马脑片以无氧无糖处理,记录脑片群峰电位(PS),部分实验以TTC染色观察脑片活性。结果OGD处理4 min为最佳损伤条件,1 h后可恢复至基础水平的(29±6)%。自由基清除剂依达拉奉(1和10 μmol·L^-1)明显增强PS波的恢复;抗炎药米诺环素(10 μmol·L^-1)和白三烯受体拮抗剂ONO-1078(1 μmol·L^-1)无显著恢复作用;阳性对照药氯胺酮也浓度依赖性促进PS恢复。结论4 min OGD为离体海马脑片缺血电生理变化的可行模型;依达拉奉对OGD脑片损伤有浓度依赖性保护作用,而米诺环素和ONO-1078未显示保护作用。     

H2 receptor mediates the protective effect of histamine on the cellular edema and viability reduction induced by oxygen- glucose deprivation in rat hippocampal slices

Aim To determine the effect of histamine on ischemia - induced cellular edema and viability reduction in rat hippocampal slices, and the involved subtypes of histamine receptor in this effect. Methods In vitro ischemic injury of hippocampal slices was induced by oxygen - glucose deprivation （ OGD）. The slice injury was determined by real - timely measuring the changes of light transmittance （LT） in CA1 region of the hippocampal slice for the cellular edema, and by detecting the product of 2, 3, 5 - trihenyherzolium chloride （ TIC）, formazan, for the slice viability.     

二氮嗪预处理大鼠海马脑片抗缺氧无糖损伤作用与激活PKC-ERK1/2通路有关

目的观察二氮嗪预处理对大鼠海马脑片缺氧无糖(oxygenglucosedeprivation,OGD)损伤的作用及PKC、ERK1/2抑制剂对其的影响。方法建立大鼠海马脑片OGD损伤模型,分别以25、50、100μmol·L-1二氮嗪灌流海马脑片30min,或分别用mitoKATP通道、PKC、ERK1/2抑制剂5HD、chelerythrine、U0126灌流30min,再用二氮嗪(100μmol·L-1)预处理,观察OGD13min、复氧1h后顺向群峰电位(orthodromicpopulationspike,OPS)的变化。结果二氮嗪预处理组(50、100μmol·L-1)OPS消失时间明显延长,复氧供糖后OPS恢复良好;二氮嗪(100μmol·L-1)预处理抗OGD损伤作用可被5HD完全取消,可被chelerythrine或U0126部分取消。结论mitoKATP通道特异性开放剂二氮嗪预处理有抗海马脑片OGD损伤作用,该效应与激活PKCERK信号通路有关。     

Depression of rat cerebral cortical neurones by H1 and H2 histamine receptor agonists

Histamine (H) and H₁ agonists-2-pyridylethylamine (PEA) adn 2-methylhistamine (2-MH) produced a greater depression of the corticospinal and unidentified rat cerebral cortical neurones than did 4-methylhistamine (4-MH), an H₂ agonist. Mepyramine antagonized teh effects of 2-MH, PEA and H, adn partially antagonized the depression induced by 4-MH. Metiamide and cimetidine, H₂ antagonists, blocked 4-MH and H but not 2-MH- and PEA-induced depression. These results indicate that H-induced depression of cortical neurones involves activation of H₁ and H₂ receptors.     

二氢石蒜碱对离体海马脑片缺氧/复氧损伤的保护作用及与NO的关系

目的:观察二氢石蒜碱对大鼠离体海马脑片脑缺氧/复氧损伤的保护作用以及与NO的关系.方法:采用大鼠离体海马脑片缺氧/复氧(H/R)损伤模型,记录群峰电位(PS)恢复幅度和恢复率及检测乳酸脱氢酶(LDH)的活性和NO含量变化.结果:与H/R模型组比较,二氢石蒜碱1×10-6和1×10-5 mol·L-1浓度显著增加H/R损伤后PS恢复幅度[(94.3±49.1)%,(71.0±41.4)%vs(14.2±34.7)%,P＜0.05]和恢复率(P＜0.05);降低孵育液中LDH的活性[(250.57±12.57),(200.74±23.71)vs(300.57±16.08)U·L-1,P＜0.05～0.01]和NO量[(0.64±0.04),(0.02±0.06)V8(4.62±0.29)mmol·L-1,P＜0.01].结论:二氢石蒜碱对大鼠离体海马脑片脑缺氧/复氧损伤有保护作用,与降低NO产生有关.     

氯胺酮的神经保护作用及其对p38蛋白磷酸化激活的影响

目的 :研究NMDA受体拮抗剂氯胺酮对大鼠海马脑片缺糖缺氧 (OGD)损伤的神经保护作用及其对p38蛋白磷酸化激活的影响。方法 :采用大鼠海马脑片体外OGD损伤模型 ,TTC染色抽提法观察氯胺酮对海马脑片OGD损伤时的神经保护作用 ,免疫印迹法研究“缺血再灌注”不同时间大鼠海马脑片中p38磷酸化激活变化情况 ,以及氯胺酮对p38磷酸化激活的影响。结果 :氯胺酮显著抑制海马脑片OGD损伤所致的A值降低 ,p38蛋白在缺血再灌注后磷酸化激活增加 ,再灌注 15min后开始上升 ,2h仍可见持续表达。氯胺酮剂量为 5、10 μmol·L-1时 ,能显著抑制p38蛋白磷酸化激活 (P <0 .0 5 )。结论 :氯胺酮具有一定的神经保护作用 ,其保护作用可能与抑制海马神经元p38蛋白的磷酸化激活有关。     

Histamine-induced excitation of spontaneously active medullary neurones in the rat brain is mediated by H2-receptors : A microiontophoretic study using H1 and H2-agonists and antagonists

The effects of histamine, applied by microiontophoresis onto spontaneously-active medullary neurones were investigated in the rat. Histamine caused current-dependent excitation of these neurones, an action that is at variance with previous studies in the cat. The nature of the receptor mediating these effects was examined using a number of agonists with differing potencies at peripheral H₁- and H₂-receptors. The precursor of histamine, l-histidine and the metabolite, N-telemethylhistamine did not mimic the effects of histamine while the H₂-agonist, 4-methylhistamine caused similar but weaker excitation. The extent of excitations produced by the H₁-agonists, 2-pyridylethylamine, 2-methylhistamine and 2-thiazolylethylamine could be related to their activity at H₂-receptors. Metiamide was ineffective in antagonising responses to histamine and related agonists as was mepyramine. The H₂-antagonist ranitidine, however, proved a good antagonist of responses to histamine and the H₁- and H₂-agonists, despite an unrelated excitatory action which may be linked to inhibition of cholinesterase. It is concluded that the excitatory effects of microiontophoretically-applied histamine and the agonists on medullary neurones in the rat is probably a result of activation of H₂-receptors.     

Study of the protective effect of calcium channel blockers against neuronal damage induced by glutamate in cultured hippocampal neurons

BackgroundThe aim of this study was to examine the putative protective effect of calcium channel blockers on hippocampal neurons in the experimental model of excitotoxic damage.MethodsSeven-day old primary dissociated cultures of rat hippocampal neural cells containing one of the following calcium channel blockers: cinnarizine, flunarizine or nimodipine were exposed to glutamate-induced injury. Quantitative assessments of neuronal injury were accomplished by measuring lactate dehydrogenase (LDH) activity in the media 24 h after exposure to glutamate and by counting and establishing the apoptotic and necrotic cells in flow cytometry with Annexin V-FITC/PI staining.ResultsIn our experiment, glutamate induced a 339% elevation of apoptotic cells and a 289% increase of necrotic cells in hippocampal neurons as compared to control cultures without drugs. In cultures containing flunarizine, glutamate-induced cell apoptosis was suppressed by 62% while necrosis showed no significant alternation. Cinnarizine exerted no anti-apoptotic effects on glutamate-injured cultured hippocampal neurons, while nimodipine intensified the apoptotic pathway of cell death and promoted an increase in the number of apoptotic neurons by 26%. When cinnarizine or nimodipine were used, the percentage of necrotic cells was significantly lower when compared with glutamate-injured cultures and it amounted to 44% and 24% for cinnarizine and nimodipine, respectively.ConclusionsThe obtained results suggest the beneficial anti-apoptotic potential of flunarizine and the anti-necrotic potential of cinnarizine against glutamate-induced death of cultured hippocampal neurons. Nimodipine can protect neurons against necrosis, but has an intensified adverse pro-apoptotic effect on cultured neurons in the experimental model of excitotoxic injury.     

DJ-1保护多巴胺能神经元抵抗过氧化氢氧化应激损伤的研究

目的 探讨DJ-1保护多巴胺能神经元免受过氧化氢(H₂O₂)损伤的机制。 方法 使用神经生长因子(NGF)将大鼠嗜铬细胞瘤细胞(PC12细胞)诱导为多巴胺能神经元模型。H₂O₂处理引起细胞氧化应激损伤模型,CCK-8试剂盒检测细胞活性,DHE染色检测细胞内ROS水平。PI / Hoechst染色检测细胞凋亡,蛋白质免疫印迹法(Western blot)检测DJ-1和TH蛋白的表达。构建DJ-1过表达载体,检测DJ-1对H₂O₂中PC12细胞的保护作用及对细胞内活性氧(ROS)的影响。RT-qPCR检测α-synuclein,p53,Bax,Bcl-2的表达变化。 结果 H₂O₂处理可显着降低PC12细胞的活性,H₂O₂处理24 h以上可引起细胞凋亡。H₂O₂处理下调DJ-1蛋白和TH蛋白的表达,并且在RNA水平上α-突触核蛋白的表达增加。另外 p53, Bax和 caspase-3表达增加, Bcl-2表达减少。DJ-1的过表达可以抑制H₂O₂引起的ROS增加,DJ-1可以维持细胞活性,减少H₂O₂的凋亡。在RNA水平抑制α-突触核蛋白,p53,bax,bcl-2的凋亡和抗凋亡基因表达变化。 结论 DJ-1能够抑制H₂O₂引起的 ROS水平升高,减少α-synuclein积累,抑制 p53,凋亡基因如 bax的表达减弱了多巴胺能神经元中H₂O₂诱导的氧化应激损伤。     

The selective histamine H1-receptor agonist 2-(3-trifluoromethylphenyl)histamine increases waking in the rat

The effects of the selective histamine H₁-receptor agonist, 2-(3-trifluoromethylphenyl)histamine, were studied in rats implanted with electrodes for chronic sleep recordings. 2-(3-Trifluoromethylphenyl)histamine (80–120 μg) injected into the left lateral ventricle increased wakefulness, whereas slow wave sleep was reduced. Pretreatment with pyrilamine (2.0 mg/kg) prevented the effects of the H₁-receptor agonist on wakefulness and slow wave sleep. Our results further support the involvement of histamine in the modulation of the waking state.