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。     

氟哌啶醇对大鼠离体海马脑片和原代神经元缺糖/缺氧和N-甲基-D-天冬氨酸损伤的保护作用

目的观察氟哌啶醇对大鼠离体海马脑片和原代神经元的缺糖/缺氧(OGD)和N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)损伤的潜在保护作用及其机制。方法海马脑片OGD以无葡萄糖的人工脑脊液中通95% N₂+5% CO₂诱导。通过测定TTC染色后形成的红色产物来分析脑片活性。结果氟哌啶醇(1和10 μmol·L^-1)抑制OGD损伤,抑制率分别为17.7%和25%,而D₂多巴胺受体拮抗剂多潘立酮无此作用。NMDA也能显著降低海马脑片及原代神经元的活性,而氟哌啶醇可抑制这一损伤作用。结论氟哌啶醇对大鼠离体海马脑片OGD和原代神经元NMDA损伤有保护作用。     

大鼠盆总神经节腺苷三磷酸释放的调节

用荧光素-荧光素酶方法测定大鼠盆总神经节腺苷三磷酸(ATP)释放. 钠通道阻断剂河豚毒素(1 μmol·L^-1)抑制电刺激诱发的盆总神经节ATP的释放. 灌流液中去除Ca^2＋并加入EGTA(1 mmol·L^-1)后消除ATP的释放. 腺苷(100 μmol·L^-1)，A₁腺苷受体激动剂环戊腺苷 (0.1 μmol·L^-1)，毒蕈碱性受体激动剂氧化震颤素(1 μmol·L^-1)和5-羟色胺(100 μmol·L^-1)减少ATP的释放. A₁腺苷受体拮抗剂8-环戊基-1，3-二丙基黄嘌呤(10 nmol·L^-1)，α₂肾上腺素受体拮抗剂育亨宾(3 μmol·L^-1)，D₂多巴胺受体拮抗剂舒必利(20 μmol·L^-1)和组胺(100 μmol·L^-1)增加ATP的释放.　结果提示， 在大鼠盆总神经节存在着作为神经递质参与突触传递的ATP释放. A₁腺苷受体，毒蕈碱性受体，α₂肾上腺素受体，D₂多巴胺受体，5-羟色胺受体及H₁组胺受体激动剂或拮抗剂可以通过节前机制影响ATP的释放.     

羟乙基葛根素对过氧化氢致牛脑微血管内皮细胞损伤的保护作用

目的研究牛脑微血管内皮细胞(BCMEC)过氧化损伤机制并探讨羟乙基葛根素对牛脑微血管内皮细胞损伤的保护作用。方法用MTT法和LDH活性检测测定BCMEC的损伤；倒置相差显微镜下一般形态学观察、透射电子显微镜超微结构观察及流式细胞术测定BCMEC凋亡变化。结果H₂O₂ (200 μmol·L^-1)损伤BCMEC后，细胞存活率下降，LDH释放增加，羟乙基葛根素和edaravone可减轻此损伤。H₂O₂ (100 μmol·L^-1)可诱导BCMEC凋亡，羟乙基葛根素 和edaravone对此有保护作用。结论羟乙基葛根素和edaravone对H₂O₂导致的BCMEC坏死和凋亡有保护作用，该作用与其抗氧化作用有关。     

脑5-HT1A/1B、α2受体及腺苷酸环化酶参与了胍丁胺的抗抑郁作用

为了在单胺受体及受体后腺苷酸环化酶(adenylate cyclase,AC)水平探讨胍丁胺(agmatine,AGM)抗抑郁作用的精细机制,采用小鼠悬尾实验和强迫游泳实验观察AGM抗抑郁行为改变。采用放射免疫方法测定大鼠前额皮层突触膜蛋白AC活性。结果表明,AGM(5～40 mg·kg^-1,ig)在小鼠悬尾实验和强迫游泳实验模型上均有显著抗抑郁活性。同时伍用β受体/5-HT_1A/1B受体阻断剂吲哚洛尔(pindolol, PIN, 20 mg·kg^-1, ip)、 α₂肾上腺素受体拮抗剂育亨宾(yohimbine, YOH, 5～10 mg·kg^-1, ip)或咪唑克生(idazoxan, IDA, 4 mg·kg^-1, ip)对AGM(40 mg·kg^-1, ig)的抗抑郁活性具有显著拮抗效应; 而β受体阻断剂普萘洛尔(propranolol, PRO, 5～20 mg·kg^-1, ip)或5-HT₃受体拮抗剂曲匹西隆(tropisetron, TRO, 5～40 mg·kg^-1, ip)对AGM(40 mg·kg^-1, ig)的抗抑郁活性无显著影响。AGM(0.1～6.4 μmol·L^-1)与大鼠前额皮层提取的突触膜共孵可剂量依赖地激活AC活性, 而PIN(1 μmol·L^-1)或YOH(0.25～1 μmol·L^-1)均显著拮抗AGM(6.4 μmol·L^-1)对AC的激活作用; 慢性给予大鼠AGM(10 mg·kg^-1, ig, bid)或氟西汀(fluoxetine, FLU, 10 mg·kg^-1, ig, bid) 2 w也显著增强大鼠前额皮层基础及Gpp(NH)p 预激活的AC活性。本研究表明, 调节脑内5-HT_1A/1B和α₂等受体功能, 并激活前额皮层AC可能是AGM抗抑郁活性的重要机制之一。     

5-单硝酸异山梨酯对兔离体隐动脉交感嘌呤能缩血管反应的影响

采用兔离体隐动脉血管环张力实验及电场刺激诱发交感嘌呤能血管收缩实验，观察5-单硝酸异山梨酯(isosorbide-5-mononitrate，ISMN)对交感嘌呤能缩血管反应的作用，并分析其作用机制。结果表明，电场刺激(电压15 V，波宽1 ms，时程1 s)诱发兔离体隐动脉(去内皮)产生血管收缩反应。该收缩反应呈频率(2～16 Hz)依赖性，可被0.1 μmol·L^-1河豚毒素(tetrodotoxin)完全抑制。α₁受体阻断药哌唑嗪(1 μmol·L^-1)对2～8 Hz电刺激诱发的血管收缩反应无影响。P2X₁受体激动药α,β-亚甲基ATP(3 μmol·L^-1)脱敏P2X₁受体，同时联合应用哌唑嗪(1 μmol·L^-1)完全抑制电刺激诱发的血管收缩反应。采用一个标本一个浓度给药时，ISMN(0.1 mmol·L^-1)显著抑制8 Hz电刺激诱发的血管收缩反应，在0.3及1.0 mmol·L^-1时ISMN显著抑制各频率电刺激诱发的血管收缩反应； 1.0 mmol·L^-1 ISMN对电刺激诱发的血管收缩反应的抑制率分别为46%(2 Hz)、 47%(4 Hz)、 34%(8 Hz)和22%(16 Hz)。ISMN(0.3及1.0 mmol·L^-1)对外源性去甲肾上腺素(0.01～100 μmol·L^-1)或腺苷三磷酸(1 mmol·L^-1)诱发的血管收缩反应无影响。以上结果提示，ISMN显著抑制电场刺激诱发的交感嘌呤能血管收缩反应，其作用机制可能是ISMN作用于交感神经末梢突触前膜抑制嘌呤能神经递质产生的血管收缩反应。     

钾通道阻断剂部分抑制三氧化二砷诱导的HeLa细胞死亡

目的研究钾通道阻滞剂对三氧化二砷诱导的HeLa细胞死亡的作用。方法采用MTT法评价HeLa细胞的存活情况，采用膜片钳技术记录HeLa细胞的电压依赖性钾电流。结果As₂O₃(5 μmol·L^-1)孵育24 h引起显著的HeLa细胞死亡，As₂O₃(5 μmol·L^-1)孵育24 h后存活的细胞表现明显的电压依赖性钾电流密度增加。+80 mV电压下，As₂O₃(5 μmol·L^-1)孵育组电流密度(61±18) pA/10 pF(n=8)明显高于对照组(38±10) pA/10 pF(n=8,P<005)。As₂O₃诱导的HeLa细胞死亡可被共同孵育钾通道阻滞剂四氨基吡啶(3 mmol·L^-1)或四乙基铵(5 mmol·L^-1)所部分抑制。3 mmol·L^-1四氨基吡啶或5 mmol·L^-1四乙基铵对HeLa细胞无明显细胞毒作用。结论As₂O₃长期处理增加HeLa细胞的电压依赖性钾电流。As₂O₃诱导的HeLa细胞死亡可被钾通道阻滞剂四氨基吡啶或四乙基铵部分抑制。     

没药甾酮对H2O2损伤PC12细胞的保护作用

探讨没药甾酮(guggulsterone)对氧化应激损伤PC12细胞的保护作用。以过氧化氢(hydrogen peroxide，H₂O₂)损伤PC12细胞为氧化应激损伤模型， 维生素E为对照， 采用四甲基偶氮唑蓝［3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide，MTT］法检测细胞增殖状况； 试剂盒检测乳酸脱氢酶(lactate dehydrogenase,LDH)及一氧化氮(nitric oxide，NO)的释放； DCFH法和Fura 2-AM法检测细胞内活性氧(reactive oxygen species，ROS)和Ca²⁺的含量； 碘化丙啶(propidium iodide，PI)染色流式细胞术(flow cytometry，FCM)检测细胞凋亡； 罗丹明123(rhodamine 123，Rh 123)染色FCM检测细胞线粒体膜电位(mitochondrial membrane protential，MMP)。结果表明， 没药甾酮(0.1～10 μmol·L^-1)可使200 μmol·L^-1 H₂O₂作用24 h后的PC12细胞生长抑制率下降； 细胞外LDH和NO， 细胞内ROS和Ca²⁺含量降低； 明显抑制200 μmol·L^-1 H₂O₂作用12 h后诱导的PC12细胞凋亡和线粒体膜电位降低作用，没药甾酮(0.1～10 μmol·L^-1)使细胞凋亡率由24.3%下降至18.4%、 15.9%、 11.8%。实验结果表明, 没药甾酮对氧化应激损伤PC12细胞具有保护作用， 其机制可能为降低细胞内ROS含量， 进而抑制LDH和NO释放， 降低细胞内Ca²⁺含量， 升高线粒体膜电位,减少细胞凋亡。     

葛根素和大豆甙元抑制[3H]氟硝西泮和体外大鼠脑膜的结合

从中药葛根中提取到两种苯二氮艹卓受体活性化合物:葛根素和大豆甙元。两种化合物在体外可抑制[3H]氟硝西泮和大鼠脑细胞膜的结合,IC₅₀值分别为18.46μmol·L^-1和15.43μmol·L^-1。大豆甙元还可抑制[³H]哌唑嗪和α1-肾上腺素受体的结合(IC₅₀值为89μmol·L^-1)。两种化合物的GABA比分别为1.11和1.12,提示两种黄酮化合物是苯二氮艹卓受体的拮抗剂或部分激动剂。Scatchardplot分析显示:两种化合物对[³H]氟硝西泮与膜结合的抑制作用是通过竞争性与非竞争性混合机制而实现的。     

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%。     

Neuroprotective effects of stearic acid against toxicity of oxygen/glucose deprivation or glutamate on rat cortical or hippocampal slices

AIM: To observe the effects of stearic acid, a long-chain saturated fatty acid consisting of 18 carbon atoms, on brain (cortical or hippocampal) slices insulted by oxygen-glucose deprivation (OGD), glutamate or sodium azide (NaN3) in vitro. METHODS: The activities of hippocampal slices were monitored by population spikes recorded in the CA1 region. In vitro injury models of brain slice were induced by 10 min of OGD, 1 mmol/L glutamate or 10 mmol/L NaN3. After 30 min of pre-incubation with stearic acid (3-30 micromol/L), brain slices (cortical or hippocampal) were subjected to OGD, glutamate or NaN3, and the tissue activities were evaluated by using the 2,3,5-triphenyltetrazolium chloride method. MK886 [5 mmol/L; a noncompetitive inhibitor of proliferator-activated receptor (PPAR-alpha)] or BADGE (bisphenol A diglycidyl ether; 100 micromol/L; an antagonist of PPAR-gamma) were tested for their effects on the neuroprotection afforded by stearic acid. RESULTS: Viability of brain slices was not changed significantly after direct incubation with stearic acid. OGD, glutamate and NaN3 injury significantly decreased the viability of brain slices. Stearic acid (3-30 micromol/L) dose-dependently protected brain slices from OGD and glutamate injury but not from NaN3 injury, and its neuroprotective effect was completely abolished by BADGE. CONCLUSION: Stearic acid can protect brain slices (cortical or hippocampal) against injury induced by OGD or glutamate. Its neuroprotective effect may be mainly mediated by the activation of PPAR-gamma.     

Protective effects of baicalin on oxygen/glucose deprivation- and NMDA-induced injuries in rat hippocampal slices

Baicalin is a flavonoid derivative from Scutellaria baicalensis Georgi with various pharmacological effects. Recently, the neuroprotective effect of baicalin was reported. To confirm this effect and explore the possible mechanism, we have investigated the protective effect of baicalin on ischaemiclike or excitotoxic injury and the activation of protein kinase C alpha (PKC(alpha)) in rat hippocampal slices. In-vitro ischaemic-like injury was induced by oxygen/glucose deprivation (OGD) and the excitotoxic injury by N-methyl-D-aspartate (NMDA). The viability and swelling of the slices were detected by triphenyltetrazolium chloride (TTC) staining and image analysis of light transmittance (LT), respectively. The translocation of PKC(alpha) was measured by immunoblotting. Baicalin was added during both injuries. Baicalin (0.1, 1, and 10 micromol L(-1)) concentration-dependently inhibited OGD-induced viability reduction and acute neuron swelling, and inhibited the increased portion of PKC(alpha) present in the membrane fraction over the total PKC(alpha). Baicalin ameliorated NMDA-induced viability reduction (not LT elevation) and inhibited the NMDA-increased membrane portion of PKC(alpha) at 1 micromol L(-1). We concluded that baicalin had a protective effect on ischaemic-like or excitotoxic injury in rat hippocampal slices, which might have been partly related to inhibition of PKC(alpha) translocation.     

5-HT2C受体对孵育海马脑片释放分泌型淀粉样前体蛋白的调节

目的考察5-HT_2C受体对孵育海马脑片释放分泌型淀粉样前体蛋白(sAPP)的调节。方法应用5-HT\,特异性5-HT_2C受体激动剂M-110及其特异性拮抗剂L-107孵育海马脑薄片,及Western blot技术和特异性的针对sAPP氨基末端的单克隆抗体22C11检测释放到孵育液中的sAPP。结果5-HT及特异性5-HT_2C受体激动剂M-110在一定的浓度范围内呈浓度依赖性地显著增加sAPP分泌;而特异性5-HT_2C受体拮抗剂L-107在一定的浓度范围内则浓度依赖性地显著抑制sAPP分泌。结论5-HT通过激活5-HT_2C受体调节孵育海马脑片分泌型淀粉样前体蛋白的释放。     

Monosialoganglioside protected ischemic rat hippocampal slices through stabilizing expression of N-methyl-D-aspartate receptor subunit

INTRODUCTION Monosialoganglioside(GM1) can alleviatecerebraledema and reduce cerebral infarct volume in in vivoanimal models[1-3], and can prevent death of culturedgranule cells in vitro from anoxia as well[4]. However,meaning of data derived from a neural preparation with-out a vasculature and mammalian systemicinteractionis limited. The direct protective effect of GM1 on is-subunits are mainly distributed in the forebrain[5], and μmol/L (control group), 0.01 μmol/L(gr…     

大鼠脑片损伤模型和新型定量评价方法的建立

目的　建立不同性质脑损伤实验模型和一种方便、快速、灵敏的定量评价方法。方法　制备大鼠皮层和海马脑片 ,检测活性后 ,分别接受缺氧缺糖 (OGD)、谷氨酸、过氧化氢 (H2 O2 )损伤 ,氯胺酮、D AP5、异丙酚预处理 ,随后与TTC溶液共同孵育 ,有机溶剂抽提 ,酶标仪测定OD4 90 值 ,同时测定孵育上清液乳酸脱氢酶 (LDH)释放率。结果　室温下恢复孵育 90min后 ,海马脑片CA1区锥体细胞层可记录到群体峰电位。随着OGD时间延长 ,皮层和海马脑片TTC染色明显降低 ,孵育上清液LDH释放率逐渐增加 ,组织损伤百分率与LDH释放率明显正相关 :皮层r =0 960 9,P <0 0 1 ;海马r =0 892 1 ,P <0 0 5。和对照组相比 ,谷氨酸 1mmol·L- 1 和H2 O2 2mmol·L- 1 明显降低脑片TTC染色。氯胺酮5μmol·L- 1 、D AP550 μmol·L- 1 、异丙酚 5μmol·L- 1 分别抑制OGD 1 0min、谷氨酸、H2 O2 损伤所致脑片TTC染色降低。结论　利用大鼠脑片建立的缺氧缺糖、谷氨酸和过氧化氢损伤实验模型 ,以及新鲜脑片与TTC溶液共同孵育 ,有机溶剂抽提、比色的定量评价方法具有方便、快速、灵敏的特点 ,能有效的用于脑缺血损伤和药效评价的研究     

西替利嗪对皮肤细胞炎症中单核趋化蛋白-1表达的干预作用

目的考察西替利嗪(CET)对皮肤细胞炎症模型中单核趋化蛋白-1(MCP-1)表达的干预作用。方法组胺(HIS)和IFN-γ刺激真皮成纤维细胞(DF)和人角质形成细胞株HaCaT细胞，采用RT-PCR和ELISA法考察两种细胞MCP-1 mRNA和蛋白表达水平。结果与对照组比较，HIS(10 μmol·L^-1)和IFN-γ(20 ng·mL^-1)组显著上调两种细胞(DF和HaCaT) MCP-1 mRNA表达，同时分别使DF的MCP-1蛋白分泌量增加3.5倍和8.4倍，对HaCaT细胞也有相似的影响趋势； CET (1和10 μmol·L^-1) 显著地抑制了它们对细胞MCP-1蛋白产生的增强作用。结论CET可能通过抑制MCP-1的表达而发挥抗皮肤炎症作用。     

Effect of levocetirizine on the contraction induced by histamine on isolated rabbit bronchioles from precision-cut lung slices

The present experiments were designed to study the effects of levocetirizine (a potent selective histamine H(1) antagonist) on the contraction induced by histamine on isolated rabbit bronchioles using the precision-cut lung slice technology. Histamine induced a concentration-dependent contraction of isolated rabbit bronchioles (pD(2) value of 5.6). Mepyramine (0.01-1 micromol/l) induced a shift to the right without any decrease in the concentration-response curve to histamine (pA(2) value of 8.2). Levocetirizine (0.03-0.1 micromol/l) induced both a shift to the right and a decrease in the maximal amplitude of the concentration-response curve to histamine (pA(2) and pD'(2) values of 7.9 and 7.0, respectively). The difference between both compounds could be explained in terms of the difference in the dissociation rate from the histamine H(1) receptor coupled to a putative low receptor reserve present in the rabbit bronchioles.     

Histamine-induced inhibition of leukotriene biosynthesis in human neutrophils: involvement of the H2 receptor and cAMP

1. Histamine is generally regarded as a pro-inflammatory mediator in diseases such as allergy and asthma. A growing number of studies, however, suggest that this autacoid is also involved in the downregulation of human polymorphonuclear leukocyte (PMN) functions and inflammatory responses through activation of the Gs-coupled histamine H(2) receptor. 2. We report here that histamine inhibits thapsigargin- and ligand (PAF and fMLP)-induced leukotriene (LT) biosynthesis in human PMN in a dose-dependent manner. 3. The suppressive effect of histamine on LT biosynthesis was abrogated by the histamine H(2) receptor antagonists cimetidine, ranitidine, and tiotidine. In contrast, the histamine H(1), H(3), and H(4) receptor antagonists used in this study were ineffective in counteracting the inhibitory effect of histamine on the biosynthesis of LT in activated human PMN. 4. The inhibition of LT biosynthesis by histamine was characterized by decreased arachidonic acid release and 5-lipoxygenase translocation to the nuclear membrane. 5. Incubation of PMN with the cAMP-dependent protein kinase (PKA) inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide prevented the inhibitory effect of histamine on LT biosynthesis, suggesting an important role for PKA in this effect of histamine on LT biosynthesis in PMN. 6. These data provide the first evidences that, similarly to adenosine and prostaglandin E(2), histamine is a potent suppressor of LT biosynthesis, and support the concept that histamine may play a dual role in the regulation of inflammation.