芍药苷对培养小鼠皮层神经元的保护作用

目的　探讨芍药苷是否促进培养皮层神经细胞的存活 ,并对抗兴奋性氨基酸海人藻酸 (KA)所致的神经损伤。方法　解剖分离 15d胚胎小鼠皮层神经细胞 ,接种于 2 4孔板中加药培养 ,台盼蓝染色细胞 ,相差显微镜及免疫组织细胞化学方法进行形态学观察。结果　①加入芍药苷 2 0 .8和 4 1.6mg·L- 1培养 4d增加神经细胞存活数量 ,降低死亡率。②加入KA 5 0 μmol·L- 1作用 30min ,神经元肿胀 ,失去折光性 ,核偏位 ,死亡率增高。预先加入芍药苷2 0 .8和 4 1.6mg·L- 1培养 4d ,可对抗KA所致的神经损伤。结论　芍药苷可增加神经细胞存活数量 ,降低死亡率 ,对抗KA所致的兴奋性神经损伤。     

L-焦谷氨酸对抗谷氨酸钠诱发的大鼠皮层神经元损伤

目的:在大鼠皮层神经元研究L-吡咯烷酮羧酸(L-PGA)对谷氨酸钠(Glu)诱发神经毒性的拮抗作用。方法:原代培养的皮层神经元取自16d龄的胎鼠,与Glu作用30分钟,24小时后测定神经元的存活及培养介质中亚硝酸盐的浓度;以Fura 2-AM为细胞内[Ca~(2 )]_i荧光探针,AR-CM-MIC阳离子测定系统测定[Ca~(2 )]_i。结果:L-PGA 10-80μmol·L~(-1)浓度依赖地抑制Glu 500μmol·L~(-1)引起的神经损伤,其IC_(50)为(41±9)μmol·L~(-1),95％可信区间:(30.3-54.7)μmol·L~(-1)。L-PGA也能浓度依赖地降低Glu引起的NO释放。L-PGA 1,3,10,30,100μmol·L~(-1)对Glu 100μmol·L~(-1)引起的[Ca~(2 )]_i升高的抑制率分别为20.5％,34.4％,47.7％,70.6％,80.4％。结论:L-PGA可能通过抑制NO形成或细胞内Ca~(2 )浓度的升高而拮抗Glu的神经毒性。     

皮质酮损伤培养的海马神经细胞并促进其电压依赖性钙内流(英文)

目的:研究皮质酮(Cor)对原代培养海马神经细胞存活和海马神经细胞电压依赖性钙通道(VDCC)的影响。方法:原代海马神经细胞存活率测定用MTT比色法。海马神经细胞上VDCC内向Ca~(2 )电流检测采用全细胞膜片箝技术。结果:Cor可浓度依赖地损伤原代海马神经细胞和皮层神经细胞,IC_(50)分别为3.2μmol·L~(-1)和85μmol·L~(-1),Cor(1μmol·L~(-1)-0.1mmol·L~(-1))喷射于海马神经细胞表面即刻显著促进电压依赖性Ca~(2 )内流,其最大升幅分别是53％,191％和84％,而且Cor诱导的钙内流增加是非浓度依赖和非电压依赖的。结论:Cor可显著促进海马神经细胞电压依赖性钙通道开放,该作用可能是Cor海马神经毒性作用的机制之一。     

三种阿尔茨海默症治疗药物促体外培养小鼠皮层神经元活性的比较

目的 :观察并比较 3种阿尔茨海默症治疗药物盐酸美金刚胺、美曲磷酯及白藜芦醇对体外培养小鼠皮层神经元活性的影响 ,为寻找作用机理更优的先导化合物提供理论依据。方法 :采用体外培养皮层神经元细胞的方法 ,解剖分离 15d胚胎小鼠皮层神经细胞 ,接种于 96孔板 ,4 8h后加药并培养72h ,相差显微镜及免疫组织细胞化学方法进行形态学观察 ,并以MTT法观察 3种药物对小鼠皮层神经元活性的影响。结果 :盐酸美金刚胺显著促进小鼠皮层神经元活性 ,并呈现出一定的量效关系 ;美曲磷酯 10pmol·L-1,0 .1,1,10nmol·L-1剂量组对皮层神经元细胞活性没有影响 ,而美曲磷酯 0 .1,1μmol·L-1剂量组显著抑制皮层神经元细胞活性 ;0 .1nmol·L-1白藜芦醇显著促进皮层神经元细胞活性 ,而白藜芦醇 0 .1,1,10 μmol·L-1剂量组显著抑制皮层神经元细胞活性。结论 :盐酸美金刚胺促小鼠皮层神经元活性较高 ,故以其为先导化合物设计药效更高的阿尔茨海默症治疗药物的方案是可行的。     

S-腺苷蛋氨酸通过降低Cav-1的表达保护KCl刺激引起的神经元损伤

目的探讨S-腺苷蛋氨酸(S-adenosylmethionine,SAM)对KCl刺激引起的原代培养的小鼠皮层神经元损伤是否有保护作用及机制。方法将原代培养1周的小鼠皮层神经元随机分为3组:正常(Control)组、KCl组、SAM-KCl组。用倒置显微镜观察各组细胞形态的变化,用q PCR、Western blot分别检测各组质膜微囊结构蛋白~(-1)(caveolin~(-1),Cav~(-1))mRNA及蛋白的表达变化。结果 0.05 mol·L~(-1)KCl作用于原代培养1周的小鼠皮层神经元12 h可使神经元产生明显的损伤,突起缩短变细,胞体变小,细胞聚团。0.02 mol·L~(-1)SAM预处理6 h可明显降低KCl引起的细胞损伤,q PCR、Western blot分析显示KCl可使原代培养1周的皮层神经元Cav~(-1) mRNA及蛋白表达增高(P<0.01),SAM可降低KCl引起的神经元Cav~(-1) mRNA及蛋白表达增高(P<0.05)。结论 SAM对KCl引起的神经元损伤有保护作用,该保护作用与其降低Cav~(-1)的表达有关。     

复智散通过细胞周期依赖性蛋白激酶5通路减轻皮层神经元Tau蛋白过度磷酸化

目的探讨复方中药复智散(FZS)能否通过抑制细胞周期依赖性蛋白激酶5(CDK5)通路,减轻Aβ_(25-351)诱导的新生鼠皮层神经元Tau蛋白过度磷酸化。方法选用24 h内新生Wistar大鼠,分离纯化皮层神经元,进行体外培养。皮层神经元在体外培养7 d后,应用20μmol·L-1Aβ_(25-351)作用于皮层神经元24 h。药物治疗组则应用FZS(20 mg·L~(-1))、CDK5抑制剂Roscovitine(15μmol·L~(-1))、钙蛋白酶(calpain)制剂Calpeptin(20μmol·L~(-1))预处理24 h,然后用20μmol·L~(-1)Aβ_(25-351)作用24 h。用Western blot检测Tau蛋白Ser396、Ser202和Thr231位点磷酸化水平和CDK5的激活蛋白p25/p35的蛋白水平;荧光酶标仪测定荧光强度来反映calpain活性;免疫沉淀法检测CDK5激酶活性。结果 20μmol·L~(-1)Aβ_(25-351)作用于皮层神经元24 h后,Tau蛋白在Ser396、Ser202、Thr231位点磷酸化水平增加,CDK5激酶活性升高,CDK5激活蛋白p25水平升高,calpain活性升高。20 mg·L-1FZS治疗组则明显抑制了Aβ_(25-351)导致的Tau蛋白在Ser396、Ser202、Thr231位点磷酸化水平增加,抑制了CDK5激酶活性、p25蛋白水平以及calpain活性升高。CDK5抑制剂roscovitine和calpain抑制剂calpeptin作为阳性对照药物也显示了抑制Tau蛋白过度磷酸化的作用。结论复智散可能通过calpain-p25/CDK5通路抑制Aβ_(25-351)导致的皮层神经元Tau蛋白过度磷酸化。     

甲基黄酮醇胺对分离的胎鼠脑细胞内游离钙浓度的影响(英文)

目的:观察甲基黄酮醇胺(MFA)对胎鼠脑细胞内游离钙浓度在静息以及激动剂存在时的作用。方法:用钙离子荧光染料Fura 2-AM负载后,测定分离的胎鼠脑细胞内游离钙浓度([Ca~(2 )]_i)及其变化。结果:在含钙1.3mmoL·L~(-1)的Hanks’液中,[Ca~(2 )]_i为197±20nmol·L~(-1)(n=44)。MFA0.15mmol·L~(-1)对静息脑细胞内钙浓度无明显影响。在细胞外钙1.3mmol·L~(-1)条件下,MFA(0.03—0.3 mmoL·L~(-1))浓度依赖性地抑制高钾去极化导致的[Ca~(2 )]_i升高,IC_(50)为0.14(95％可信限:0.05—0.42)mmoL·L~(-1)。在较高浓度时,MFA(0.15—0.3mmoL·L~(-1))也可抑制谷氨酸兴奋所引起的[Ca~(2 )]_i,IC_(50)为0.20(95％可信限:0.01—3.40)mmoL·L~(-1)。结论:MFA抑制高钾去极化引起的[Ca~(2 )]_i升高,在较高浓度时也拮抗谷氨酸兴奋所致的[Ca~(2 )]_i升高。     

姜黄素对Aβ_(1-42)诱导的细胞损伤和线粒体途径细胞凋亡的抑制作用

目的探讨姜黄素对Aβ_(1-42)诱导的细胞损伤和凋亡的抑制作用。方法采用体外培养的人神经母细胞瘤SH-SY5Y细胞,分为溶剂对照组、Aβ_(1-42)10μmol·L~(-1)损伤组、Aβ_(1-42)10μmol·L~(-1)+姜黄素1,5和10μmol·L~(-1)保护组及姜黄素10μmol·L~(-1)对照组;噻唑蓝(MTT)法测定细胞存活率,酶活性法检测细胞培养液中乳酸脱氢酶(LDH)的含量以考察细胞损伤程度;AnnexinⅤ-FITC/PI染色法测定细胞凋亡;JC-1染色法检测线粒体膜电位变化;比色法测定胱天蛋白酶9和胱天蛋白酶3的活性;Western蛋白印迹法检测胱天蛋白酶3表达。结果与溶剂对照组比,Aβ_(1-42)10μmol·L~(-1)损伤组细胞存活率显著降低(P<0.01)。与Aβ_(1-42)10μmol·L~(-1)损伤组比较,姜黄素5和10μmol·L~(-1)缓解了Aβ_(1-42)诱导的细胞存活率下降(P<0.05),降低了Aβ_(1-42)诱导的乳酸脱氢酶释放水平(P<0.01)和细胞早期及晚期凋亡率(P<0.01)。姜黄素抑制了Aβ_(1-42)诱导的细胞线粒体膜电位去极化作用(P<0.01);姜黄素1~10μmol·L~(-1)抑制了Aβ_(1-42)诱导的胱天蛋白酶9及胱天蛋白酶3级联激活作用,且呈浓度依赖性(r=0.990,P<0.01;r=0.996,P<0.01)。姜黄素10μmol·L~(-1)对照组以上指标与溶剂对照组无明显差异。结论姜黄素可能通过升高线粒体膜电位、降低胱天蛋白酶活性抑制Aβ_(1-42)诱导的细胞损伤和线粒体途径细胞凋亡。     

氯化锂对冈田酸所致SK-N-SH神经元突触萎缩的保护作用

目的考察氯化锂(LiCl)对蛋白磷酸酶抑制剂冈田酸(OA)诱导的SK-N-SH细胞分化神经元损伤的保护作用和tau蛋白Ser-~(262)位点磷酸化水平的影响。方法利用全反式维甲酸(ATRA)诱导SK-N-SH细胞分化为成熟的神经元细胞;采用OA诱导成熟神经元细胞建立AD模型;采用磺酰罗丹明B(SRB)比色法考察LiCl对成熟的神经元细胞增殖的抑制作用;Giemsa染色观察SK-N-SH细胞形态学变化;并采用Image-Proplus软件测定神经元细胞的突触长度;采用Western blot检测synaptophysin蛋白和tau蛋白Ser-~(262)位点磷酸化水平。结果 10μmol·L~(-1)ATRA连续处理7 d,可诱导SK-N-SH细胞突触生长和synaptophysin蛋白表达等典型分化神经元的特征。20~100 nmol·L~(-1)OA作用于分化神经元,可浓度和时间依赖性抑制细胞增殖,同时致分化神经元突触萎缩,tau蛋白Ser-~(262)位点磷酸化水平也明显升高。10 mmol·L~(-1)LiCl预处理可维持synaptophysin蛋白高表达,抑制tau蛋白Ser-~(262)位点磷酸化水平(P<0.01)。结论LiCl能够改善OA所致分化神经元的突触损伤,并伴随着synaptophysin表达的升高、tau蛋白Ser-~(262)位点异常磷酸化水平的降低。     

银杏提取物对过氧化氢损伤的培养大鼠心肌细胞的保护作用

目的:探讨银杏叶提取物(GbE)对过氧化氢损伤的培养心肌细胞的保护作用及其机制。方法:比色法测定乳酸脱氢酶活性;戊巴比妥酸法测定细胞内脂质过氧化物含量;透射电镜下观察细胞超微结构。结果:过氧化氢导致心肌细胞LDH释放从(2166±247)U·L~(-1)增至(5180±648)U·L~(-1),MDA含量从每10~6细胞(3.5±0.2)nmol增至(7.2±0.4)nmol;心肌细胞超微结构受到严重损伤。加GbE使LDH释放从(5180±648)U·L~(-1)降至(3496±386)U·L~(-1);MDA生成由每10~6细胞(7.2±0.4)nmol降至(4.8±0.9)nmol并减轻心肌超微结构的损伤。结论:GbE通过清除氧自由基保护过氧化氢损伤的心肌细胞。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.