二甲基精氨酸二甲胺水解酶与心血管疾病

二甲基精氨酸二甲胺水解酶(DDAH)是一种胞浆蛋白酶,包括DDAH1和DDAH2两种亚型,能特异性水解内源性一氧化氮合酶(NOS)抑制物非对称二甲基精氨酸而上调NOS活性。DDAH与NOS活性之间的相互作用在调节NO生成和血管内皮功能中起重要作用。DDAH还参与血管新生与细胞分化的调节,其活性变化与动脉粥样硬化等多种心血管疾病的发生发展密切相关,可能是一个新的心血管疾病相关蛋白和药物防治靶点。     

甲基苯丙胺所致的大鼠神经毒性损伤及nNOS抑制剂的保护作用

目的探讨神经元型一氧化氮合酶(nNOS)在甲基苯丙胺(METH)所致大鼠中枢神经系统氧化应激损伤中的作用机制。方法建立METH中毒动物模型,并给予nNOS抑制剂7硝基吲唑(7-NI)预处理,观察各组间动物行为学的变化,并检测nNOS表达、多巴胺(DA)含量、硝基化蛋白表达及凋亡等指标。结果 METH组大鼠纹状体中nNOS蛋白表达水平明显升高,而METH+7-NI组nNOS表达水平较METH组明显降低。METH组DA明显降低(P<0.01),而METH+7-NI组、7-NI组与对照组比较,DA无差异(P>0.05)。METH组硝基化蛋白含量明显升高(P<0.01),而METH+7-NI组硝基化蛋白表达较METH组明显降低(P<0.05)。METH组与对照组相比,凋亡细胞数明显升高(P<0.01),而7-NI对凋亡的增高表现出明显的保护作用(与METH组相比P<0.01)。METH组和METH+7-NI组的动物刻板行为评分均明显高于7-NI和对照组(P<0.01),METH、METH+7-NI组间差异无显著性(P>0.05)。结论METH可导致大鼠大脑纹状体nNOS蛋白表达水平增高、DA含量的降低、硝基化蛋白水平升高及细胞凋亡增多等多种神经毒性表现,7-NI能部分减轻其神经毒性作用。METH明显增加大鼠的不自主刻板运动,而应用7-NI预处理后,对刻板行为的改善并不明显。     

吡咯烷二硫代氨基甲酸盐对糖尿病大鼠血管内皮功能不全的影响及其机制

目的探讨吡咯烷二硫代氨基甲酸盐(PDTC)对糖尿病大鼠血管内皮依赖性舒张功能损害的保护作用及其机制。方法雄性SD大鼠一次性ip给予链脲佐菌素60 mg·kg-1制备糖尿病模型,通过饮水中给予PDTC 10 mg·kg-1,连续治疗8周。检测血糖、血脂和血清内源性一氧化氮合酶(NOS)抑制物非对称性二甲基精氨酸(ADMA)浓度。用含有人二甲基精氨酸二甲胺水解酶2(h DDAH2)基因的重组腺病毒(Ad5CMV-h DDAH2)体外感染糖尿病大鼠血管环,分别检测感染前后血管环对乙酰胆碱诱导的最大舒张反应(Emax)、半数有效量(EC50)及血管组织DDAH活性。结果与正常组比较,糖尿病大鼠血糖明显升高,血清ADMA浓度从正常组的(1.14±0.26)μmol·L-1升至(2.18±0.52)μmol·L-1(P<0.01);血管组织DDAH活性也从正常组的(0.10±0.02)U·g-1蛋白降至(0.05±0.01)U·g-1蛋白(P<0.01);血管内皮依赖性舒张功能损伤,表现为Emax由正常组的(93.6±4.4)%降至(50.8±4.9)%(P<0.01),EC50由正常组的(88±22)nmol·L-1升至(240±45)nmol·L-1(P<0.01)。PDTC治疗降低血糖和血清ADMA浓度分别至(13.2±3.5)mmol·L-1和(1.40±0.25)μmol·L-1(P<0.01),增加血管DDAH活性至(0.08±0.02)U·g-1蛋白(P<0.01),改善内皮依赖性血管舒张功能,使Emax增至(84.6±4.5)%,EC50降至(134±27)nmol·L-1(P<0.01)。糖尿病大鼠血管转染DDAH2基因后,血管DDAH活性及Emax和EC50的变化与PDTC治疗组相似。结论 PDTC对糖尿病大鼠血管内皮依赖性舒张功能具有明显的保护作用,其机制可能与上调血管DDAH活性,降低内源性NOS抑制物ADMA蓄积有关。     

RGS4过表达对甲基苯丙胺依赖大鼠纹状体mGluR5信号传导通路相关蛋白表达的影响

目的通过建立大鼠甲基苯丙胺(METH)依赖条件位置偏爱模型(CPP),研究脑纹状体内G蛋白信号调节因子4(RGS4)过表达引起的代谢型谷氨酸受体5(m GluR5)信号传导通路相关蛋白表达变化,以及对大鼠CPP行为的影响。方法将大鼠随机分为正常组、生理盐水(NS)组、METH组、Ad5-RGS4-EGFP组、Ad5-EGFP组。正常组不做处理,其余各组脑纹状体分别立体定位注射磷酸盐缓冲液(PBS)、PBS、过表达腺病毒载体Ad5-RGS4-EGFP和阴性对照腺病毒载体Ad5-EGFP,分析各组大鼠CPP行为,Western blot检测脑纹状体内RGS4、m GluR5、Gαq、PLCβ1等蛋白的表达。结果Ad5-RGS4-EGFP组CPP差值低于METH组和Ad5-EGFP组(P <0. 05),高于NS组(P <0. 05)。Ad5-RGS4-EGFP组RGS4蛋白表达均不同程度高于其余4组(P <0. 05,P <0. 01),m GluR5、Gαq表达低于METH组和Ad5-EGFP组(P<0. 05),略高于正常组和NS组(P> 0. 05),PLCβ1表达略有变化,但差异无显著性。结论纹状体内RGS4过表达可明显缓解METH成瘾性大鼠CPP行为,机制可能与RGS4过表达后,负性调控m GluR5介导的Gαq及PLCβ1信号传导通路相关。     

卡维地洛对氧化低密度脂蛋白诱导血管内皮细胞ADMA-DDAH系统的影响

目的 观察卡维地洛对氧化低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVECs)二甲基精氨酸-二甲基赖氨酸水解酶(DDAH)活性及表达的影响,以探讨卡维地洛对内源性一氧化氮合酶抑制物不对称二甲精氨酸(ADMA)代谢机制的影响.方法 采用改良的Jaffe法培养原代人脐静脉内皮细胞(HUVECs),取生长良好的3～6代HUVECs用于实验,分为(1)空白对照组:加DMEM培养液;(2)ox-LDL组:加入ox-LDL(100 mg/L,150 mg/L);(3)ox-LDL 卡维地洛组:同时加入150 mg/L ox-LDL及卡维地洛(10 μmol/L)共孵24h后,检测上清液中NO、NOS活性、ADMA含量、L-胍氨酸(L-cit)浓度,采用Western blotting测定细胞裂解液中二甲基精氨酸-二甲基赖氨酸水解酶(DDAH)的蛋白表达.结果 ox-LDL条件培养下,内皮细胞的代谢产物ADMA、ET的量均较空白对照组高,而NO的量及NOS的活性减少;反应DDAH酶活性的L-cit浓度显著降低,且有浓度依赖性,而DDAH的表达无明显变化.卡维地洛干预后,ADMA、ET的量较ox-LDL组降低,NOS活性及NO增加,L-cit浓度明显升高.结论 ox-LDL诱导下,内皮损伤ADMA的增加与DDAH的活性减弱有关,而与DDAH的表达无关.卡维地洛通过增加DDAH活性促进ADMA代谢,使NOS活性增高,抑制ox-LDL对内皮功能的损伤.     

N-乙酰半胱氨酸对甲基苯丙胺中毒大鼠行为改变的影响

目的:探讨N-乙酰半胱氨酸(NAC)对甲基苯丙胺(METH)引起中毒大鼠模型行为改变的保护性作用机制。方法:制备大鼠中毒模型,在METH注射前30min腹腔注射NAC,应用二氯荧光乙酰乙酸盐(DCFH)作为荧光指标检测大鼠纹状体ROS的含量,以紫外分光光度计检测NOS的活性,以Sams-Dodd的方法给大鼠刻板行为评分,并计算不同组大鼠评分之间的差异。结果:NAC预处理能降低纹状体内ROS的含量(P<0.001)及NOS的活性(P<0.001),减轻METH中毒大鼠精神行为改变,降低了中毒大鼠的刻板行为评分(P<0.001)。结论:NAC可能通过逆转METH诱导大鼠纹状体区的氧化失衡状态,减轻氧化应激诱导的精神行为异常改变,产生保护性作用。     

甲基苯丙胺致大鼠神经毒性及纹状体硝化作用的改变

目的探讨甲基苯丙胺(methamphetamine,MA)所导致的大鼠神经毒性及纹状体硝化作用的改变。方法建立MA给药模型,对照组给予等量生理盐水;观察两组大鼠的行为学改变,体温变化,纹状体中NO含量,NOS活性和蛋白质硝化水平的变化情况。结果MA组大鼠刻板行为评分和体温均高于对照组(P<0.01);与对照组相比,MA组大鼠纹状体NO含量及NOS活性增加(P<0.01);纹状体蛋白硝化水平明显增加(P<0.01),差异有显著性。结论MA可导致大鼠行为学改变和体温变化,大鼠脑组织NO含量、NOS活性及蛋白硝化水平的增高可能是MA中枢神经系统损伤的重要机制。     

反复自然流产患者血清及绒毛组织细胞因子研究

目的　对比观察反复自然流产组 (RSA)与正常妊娠人工流产组患者血清中一氧化氮 (NO)含量、一氧化氮合酶 (NOS)活性和血管内皮生长因子 (VEGF)含量及绒毛组织中NOS活性、P5 3蛋白及bcl 2蛋白的表达水平 ,为RSA的防治提供依据。方法　 3 3例RSA患者及 3 0例正常妊娠人工流产者血清NO含量、血清及绒毛组织中NOS活性检测 ,均采用比色法进行 ;血清VEGF含量检测采用ELISA方法 ;绒毛组织中P5 3蛋白及bcl 2蛋白的表达水平采用流式细胞仪检测。结果　RSA组血清NO含量较正常妊娠人工流产组升高 ( P <0 .0 5 ) ;血清NOS活性较对照组升高 ( P <0 .0 1) ;绒毛组织中NOS活性较对照组升高 (P <0 .0 1) ;血清VEGF含量较对照组升高 (P <0 .0 1) ;绒毛组织中P5 3蛋白表达 ,较对照组升高 (P <0 .0 1) ;绒毛组织中bcl 2蛋白表达 ,较对照组降低 (P <0 .0 1)。结论　RSA组患者血清中NO含量、NOS活性及VEGF含量和绒毛组织中NOS活性、P5 3蛋白表达量均明显升高 ,bcl 2蛋白表达量明显减低 ,均可导致胎盘细胞凋亡增强 ,可能与RSA的发病有关     

不同时期高血压大鼠心肌组织eNOS及Ser1179活性变化

目的 通过观察不同时期高血压大鼠心肌组织内皮型一氧化氮合酶（eNOS）表达及其相关调控位点Ser1179磷酸化水平、NOS活性和NO产量变化,来探讨血压变化对心肌组织eNOS活性的影响.方法 将24只SD大鼠随机分为四组,假手术2W、4W组和高血压2W、4W组,每组6只,采用双肾双狭（2K2C）复制肾血管性高血压大鼠模型.鼠尾测压法测定大鼠血压;Western Blot分别检测大鼠心肌组织eNOS、P-NOS（P-Ser1179）蛋白表达;心肌组织NO测定采用硝酸还原酶法.结果 与假手术组相比,术后2周时,RH大鼠心肌组织eNOS蛋白表达及Ser-1179磷酸化水平无明显变化（P＜0.05）;术后4周时,RH大鼠心肌组织eNOS蛋白表达、Serl179磷酸化水平显著提高（P＜0.01）.结论 肾血管性高血压大鼠心肌组织存在着eNOS蛋白表达及活性变化,eNOS具有重要的代偿调节作用,可能与血压升高刺激Serl179的磷酸化水平升高,上调NOS活性有关.     

沉默Fis1基因对METH诱导体外培养SH-SY5Y细胞增殖能力、线粒体膜电位和超微结构的影响

目的评估线粒体分裂蛋白1(Fis1)在甲基苯丙胺(METH)诱导人神经母细胞瘤细胞(SH-SY5Y细胞)损伤中的作用。方法采用成组设计方法,将体外培养SH-SY5Y细胞分为不同组别:未沉默组、沉默阴性组和沉默组,不同浓度的METH诱导各组SH-SY5Y细胞24 h。利用Western blot检测Fis1蛋白表达水平,使用CCK-8细胞毒性增殖实验分析METH对SH-SY5Y细胞增殖能力的影响,利用线粒体膜电位检测试剂盒(JC-1)检测METH对SH-SY5Y细胞MMP水平的影响,使用透射电镜观察METH对SH-SY5Y细胞线粒体超微结构的影响。结果未沉默组、沉默阴性组和沉默组中,与对照组比较,各组组内随METH诱导SH-SY5Y细胞的浓度升高,Fis1蛋白表达水平升高(P<0.05)、增殖能力减弱(P<0.05)、MMP水平降低(P<0.05);与未沉默组和沉默阴性组相同浓度比较,沉默组SH-SY5Y细胞Fis1蛋白表达水平降低(P <0. 05),增殖能力增强(P <0. 05),MMP水平升高(P <0. 05)。组内与对照组比较,2. 0mmol·L~(-1)METH诱导未沉默组、沉默阴性组和沉默组,透射电镜观察见线粒体小球状结构增多(P <0. 01)。结论 Fis1可能在METH诱导体外培养SH-SY5Y细胞损伤中起关键作用。     

Quantitative in vivo and in vitro sex differences in artemisinin metabolism in rat

1. The pharmacokinetics of the antimalarial compound artemisinin were compared in the male and female Sprague-Dawley rat after single dose i.v. (20 mg.kg) or i.p. (50 mg.kg) administration of an emulsion formulation. 2. Plasma clearance of artemisinin was 12.0 (95% confidence interval: 10.4, 13.0) l.h. kg in the male rat and 10.6 (95% CI: 7.5, 15.0) l.h. kg in the female rat suggesting high hepatic extraction in combination with erythrocyte uptake or clearance. Artemisinin half-life was 0.5 h after both routes of administration in both sexes. Values for plasma clearance and half-lives did not statistically differ between the sexes. 3. After i.p. administration artemisinin AUCs were 2-fold higher in the female compared with male rat (p 0.001). Artemisinin disappearance was 3.9-fold greater in microsomes from male compared with female livers and it was inhibited in male microsomes by goat or rabbit serum containing antibodies against CYP2C11 and CYP3A2 but not CYP2B1 or CYP2E1. 4. The unbound fraction of artemisinin in plasma was lower (p 0.001) in plasma obtained from the male (8.8 2.0%) compared with the female rat (11.7 2.2%). 5. The possibility of a marked sex difference, dependent on the route of administration, has to be taken into account in the design and interpretation of toxicological studies of artemisinin in this species.     

Theophylline kinetics in peripheral tissues in vivo in humans

Several biochemical and cellular effects have been described for methylxanthines under in vitro conditions. However, it is unknown, whether threshold concentrations required to exert these effects are attained in target tissues in vivo. We therefore employed the microdialysis technique for measuring theophylline concentrations in peripheral tissues under in vivo conditions.Following in vitro and in vivo calibration, microdialysis probes were inserted into the medial vastus muscle and into the periumbilical subcutaneous adipose layer of healthy volunteers. Following single oral dose administration of 300 mg or i.v. infusion of 240 mg theophylline, in vivo time courses of theophylline concentrations were monitored in tissues and plasma. Major pharmacokinetic parameters (c_max, t_max, AUC) were calculated for plasma and tissue time courses. The mean AUC_tissue /AUC_plasma-ratio was 0.56 (p.o.) and 0.55 (i.v.) for muscle and 0.55 (p.o.) and 0.72 (i.v.) for subcutaneous adipose tissue.We conclude that microdialysis provides important information on the distribution and the tissue pharmacokinetics of theophylline.Abbreviations FPIA Fluorescence polarisation immuno assay - AUC Area under the curve - t_max Time to peak concentration - c_max Peak concentration     

Quantitative in vivo and in vitro sex differences in artemisinin metabolism in rat

1. The pharmacokinetics of the antimalarial compound artemisinin were compared in the male and female Sprague-Dawley rat after single dose i.v. (20 mg x kg(-1)) or i.p. (50 mg x kg(-1)) administration of an emulsion formulation. 2. Plasma clearance of artemisinin was 12.0 (95% confidence interval: 10.4, 13.0) 1 x h(-1) x kg(-1) in the male rat and 10.6 (95% CI: 7.5, 15.0) 1 x h(-1) x kg(-1) in the female rat suggesting high hepatic extraction in combination with erythrocyte uptake or clearance. Artemisinin half-life was approximately 0.5 h after both routes of administration in both sexes. Values for plasma clearance and half-lives did not statistically differ between the sexes. 3. After i.p. administration artemisinin AUCs were 2-fold higher in the female compared with male rat (p < 0.001). Artemisinin disappearance was 3.9-fold greater in microsomes from male compared with female livers and it was inhibited in male microsomes by goat or rabbit serum containing antibodies against CYP2C11 and CYP3A2 but not CYP2B1 or CYP2E1. 4. The unbound fraction of artemisinin in plasma was lower (p < 0.001) in plasma obtained from the male (8.8 +/- 2.0%) compared with the female rat (11.7 +/- 2.2%). 5. The possibility of a marked sex difference, dependent on the route of administration, has to be taken into account in the design and interpretation of toxicological studies of artemisinin in this species.     

Interindividual variability in metabolic activation in humans in vivo

In assessing interindividual variability in metabolic activation, the toxic metabolite is often too unstable for conventional analysis. Possible alternatives include a stable product of the reactive metabolite e.g. cysteinyl derivatives of N-acetyl-4-benzoquinoneimine, the toxic metabolite of paracetamol, adducts with DNA or protein, and indirect measurement of the activity of the enzyme(s) producing the active metabolite. An example of the last approach is the use of furafylline, a highly specific inhibitor of human CYP1A2, to determine the extent of the metabolic activation of the cooked food mutagens PhIP and MeIQx. The extent of inhibition, determined from levels of unchanged amine in urine, is an indirect measure of the activity of the activation pathway. Further refinement of this approach, allied to improved measures of the biological process of interest should prove of value in evaluating interindividual variability and its role in the risk assessment process.     

休克时血中氧自由基的变化

本实验测定10名休克患者血浆和红细胞的丙二醛(MDA)、血浆总抗的氧化活性(AOA)的含量。结果表明:休克病人红细胞膜和血浆 MDA 含量(4.298±0.722;5.348±0.834)与对照组(3.235±0.682;4.356±1.081)比较明显增高(P<0.05);血浆 AOA(39.65±7.858)与对照组(48.21±10.81)比较明显降低(P<0.01)。提示:休克时,患者机体内自由基反应增强是引起组织细胞损伤的原因之一。     

Hyperkalaemia in patients in hospital

A survey of all laboratory blood specimens with a plasma potassium concentration greater than or equal to 5.5 mmol/L was conducted over a three month period. Of 331 specimens with hyperkalaemia, 71 were excluded because the specimens was haemolysed, old or contaminated. The laboratory served a population of 348,561 and during this time measured the plasma potassium on 25,016 occasions. Sixty-six outpatients and 20 neonates were not evaluated. The survey was undertaken on 86 of 102 inpatients (46 males), 48 of whom were over 66 years of age. Fifty-seven patients were admitted under a medical service and 29 under a surgical service. Fifty-nine had a single episode of hyperkalaemia. Thirty-two underwent a surgical procedure. The commonest contributing factor was impaired renal function which was present in 71 (83%) patients. Although a definitive causative role for drugs could be identified in only five patients, in 52 (60%) patients drugs were a contributing factor (potassium supplements 24, ACE inhibitors 16, nonsteroidal antiinflammatory drugs 12). Thirty-five of the 86 (41%) patients died during their hospital admission. Nineteen of the 35 deaths occurred within three days of the hyperkalaemia being recorded. A normal plasma potassium was eventually documented in 50 of the 86 patients. Of the remaining 36 patients, 25 (69%) subsequently died. In general the treatment of patients with hyperkalaemia focused on identifying and treating the underlying cause. Hyperkalaemia must always be considered seriously and regard given to the overall clinical status of the patient, with particular attention to drug therapy, renal and cardiac function, acid base status and the possibility of sepsis.     

Trichinellosis in immigrants in Switzerland

We describe a case of trichinellosis diagnosed at the Division of Infectious Diseases, Hospital of Lugano, in January 2009. This case was associated with a cluster of cases and was traced to the consumption of contaminated meat after a wild boar hunt in Bosnia.