蛋白酶激活受体-2在大鼠体内体外脑缺血模型中的作用研究

目的研究蛋白酶激活受体-2(PAR-2)在大鼠体内外脑缺血模型中的作用。方法以PAR-2激活肽(AP)和拮抗剂(Anta)为工具药,使用大脑中动脉阻塞和氧糖剥夺的方法分别建立大鼠和大鼠胚胎脑皮质神经元的缺血模型,观察PAR-2在缺血损伤中的作用及表达变化。用肌动描记器记录PAR-2对正常或来自脑缺血大鼠的大脑中动脉(MCA)血管张力的影响。结果蛋白质印迹法表明PAR-2在缺血侧大脑皮质中表达增多,缺血前给予PAR-2 AP(SLIGRL-NH2)显著改善了体内和体外模型缺血导致的损伤,且能够被PAR-2 Anta(ENMD-1068)所抑制。PAR-2 AP能够使MCA产生一氧化氮依赖性舒张,但是不能被ENMD-1068所抑制。结论 PAR-2有望作为预防性治疗脑缺血疾病的靶点进行研究,ENMD-1068能够抑制SLIGRL-NH2对脑缺血的保护作用。     

文冠果壳苷对大鼠大脑中动脉缺血再灌注损伤的改善作用及机制初探

目的考察文冠果壳苷对大鼠大脑中动脉缺血再灌注损伤的改善作用,并从改善突触功能的角度,探讨其作用机制。方法利用大脑中动脉阻塞法(MCAO)制备大鼠局灶性脑缺血再灌注损伤模型,TTC染色法计算脑梗死面积,HE染色法观察海马CA1区神经细胞病理形态改变,透射电镜观察大脑皮层缺血半暗带区神经元及突触超微结构改变,Western blotting检测突触相关蛋白SYP、PSD95及GAP43的表达。结果文冠果壳苷显著改善模型大鼠神经功能缺失症状,减少脑梗死面积,改善海马CA1区神经细胞的病理改变,并改善脑缺血半暗带区神经元及突触超微结构的损伤,增加突触相关蛋白SYP、PSD95及GAP43的表达。结论文冠果壳苷可显著改善大鼠大脑中动脉缺血再灌注损伤,其机制可能与促进突触重塑和/或减轻突触结构与功能的损伤有关。     

复方蒲黄颗粒对局灶性脑缺血再灌注大鼠神经元凋亡及Caspase-3 mRNA表达的影响

目的:观察复方蒲黄颗粒(PH)对局灶性脑缺血再灌注大鼠神经元凋亡的影响,并探讨其作用机制。方法:采用大脑中动脉线栓法建立局灶性脑缺血再灌注大鼠模型。75只大鼠分为假手术、模型和PH高、中、低剂量组。采用Nissl染色观察神经元形态变化,TUNEL法检测神经元凋亡,原位杂交检测Caspase-3mRNA的表达。结果:PH高、中剂量组少量细胞核固缩深染,细胞丢失、凋亡细胞数及Caspase-3mRNA阳性细胞数均显著减少。结论:PH可下调Caspase-3的表达,从而抑制神经元凋亡,对脑缺血再灌注损伤有保护作用。     

新型P2Y样G蛋白偶联受体GPR17在脑缺血损伤中的作用

目的探讨新型P2Y样G蛋白偶联受体GPR17对脑缺血及缺氧缺糖(OGD)诱导皮层混合培养细胞中神经元损伤及小胶质细胞激活的影响。方法①以线栓法制备大鼠局灶性脑缺血模型,采用免疫组织化学、Western blotting、RT-PCR以及免疫荧光等方法观察脑内GPR17的时空表达及细胞分布特点。大鼠侧脑室埋管给予GPR17 siRNA靶向沉默脑内GPR17表达,观察其对脑缺血急、慢性期神经元损伤和小胶质细胞激活的影响。②以OGD诱导大鼠皮层混合培养细胞的缺血性损伤,以GPR17 siRNA靶向沉默GPR17的表达,观察其对OGD诱导的原代皮层混合培养细胞中神经元损伤和小胶质细胞激活的影响。结果①缺血中心区,GPR17 mRNA及蛋白水平在再灌注24 h和7,14 d表达上调;缺血周边区,再灌注7,14 d表达上调。在正常大鼠脑组织,GPR17主要表达于神经元、少突胶质细胞。在缺血中心区,再灌注24 h GPR17主要表达于损伤的神经元、小胶质细胞和少突胶质细胞,再灌注14 d主要表达于增生激活的小胶质细胞,部分表达于少突胶质细胞;而在缺血周边区,再灌注24 h以及14 d,GPR17主要表达于神经元、小胶质细胞和少突胶质细胞。星形胶质细胞不表达GPR17。大鼠侧脑室给予GPR17 siRNA成功抑制脑内GPR17表达,显著改善再灌注24 h神经症状、减少脑梗死体积以及神经元损伤;同样,也改善再灌注14 d的脑萎缩和周边区的神经元损伤,并显著抑制小胶质细胞的增生激活。②大鼠原代皮层混合培养细胞中,OGD 1 h恢复24 h(OGD/R)诱导细胞活性降低,LDH释放增加,PI染色结果显示细胞坏死增加,以神经元死亡为主。GPR17 siRNA处理后减轻OGD/R诱导的细胞活性下降以及LDH释放,并减轻细胞坏死,以减轻神经元死亡为主;GPR17siRNA处理后能够改善小胶质细胞激活的形态变化。结论大鼠局灶性脑缺血后,脑内GPR17表达上调,介导缺血后急性神经元损伤以及亚急性/慢性期的小胶质细胞激活。GPR17还介导OGD诱导的大鼠皮层混合培养细胞中的神经元损伤和小胶质细胞激活。     

脑缺血再灌注后CIDE-B表达与神经元凋亡的关系

目的探讨大鼠脑缺血/再灌注损伤后细胞死亡DNA片段裂解因子45样因子B(CIDE-B)表达与海马神经元凋亡的关系。方法成年健康雄性Wistar大鼠,应用线栓法建立大鼠大脑中动脉缺血再灌注模型,TUNEL法染色观察海马神经元凋亡,免疫组化法和Western blot法检测海马神经元CIDE-B蛋白表达,RT-PCR检测CIDE-B mRNA表达。结果与假手术组比较,脑缺血2h再灌注6h,1d,3d,7d,14d,凋亡神经元显著增加(P0.01),CIDE-B mRNA和CIDE-B蛋白表达明显增强加(P0.01);至缺血2h再灌注28d神经元凋亡数量显著较少,CIDE-B mRNA和蛋白表达明显减弱(P0.01),细胞凋亡与CIDE-B基因表达的时相一致。结论脑缺血再灌注损伤后CIDE-B表达与神经元凋亡呈时相依赖性。     

牛蒡子复方制剂对大鼠脑缺血再灌注后Caspase-3表达的影响

目的：探讨牛蒡子复方制剂（WWZ）对大鼠脑缺血再灌注后Caspase-3表达的影响。方法：采用改良线栓法制作右侧大脑中动脉缺血再灌注模型,运用Zea-Longa法进行神经病学评分,用光镜观察脑组织形态学改变,用免疫组化法观察其对Caspase-3蛋白表达的影响。结果：WWZ治疗组脑组织变性坏死程度轻,神经功能缺损评分显著减少,Caspase-3阳性细胞减少（P〈0.01）。结论：WWZ能降低神经功能缺失评分、抑制Caspase-3的激活,提示对大鼠局灶性脑缺血再灌注损伤有保护作用,其脑保护作用可能与抑制缺血区Caspase-3蛋白表达从而抑制细胞凋亡有关。     

羟乙葛根素对大鼠局灶性脑缺血再灌注损伤细胞凋亡及p53表达的影响

目的观察异黄酮类羟乙葛根素在大鼠局灶性脑缺血再灌注损伤时的抗凋亡作用。方法利用大鼠大脑中动脉栓线阻断法制备局灶性脑缺血再灌注损伤模型 ,利用病理学检查、免疫组织化学方法观察羟乙葛根素对缺血再灌注大鼠的脑组织细胞凋亡及p5 3表达的影响。结果羟乙葛根素能改善局灶性脑缺血再灌注损伤的病理学改变 ,减轻细胞凋亡的程度 ,抑制p5 3的表达。结论羟乙葛根素对大鼠局灶性脑缺血再灌注损伤具有保护作用 ,其作用机制可能与羟乙葛根素抗细胞凋亡 ,抑制p5 3表达有关。     

大鼠面神经缺血损伤后PAR-1、Src及p-Src的表达变化及意义

目的研究大鼠面神经缺血损伤后面神经元中PAR-1、Src及Src磷酸化（p-Src）的表达变化及意义。方法利用外科手术方法阻断大鼠鼓室段岩动脉导致面神经缺血损伤后,取60只SD大鼠,通过腹腔注射水蛭素,运用免疫印迹、RT-PCR方法对假手术组（SH）、岩动脉阻断损伤组（PAI）、PAI＋生理盐水组（NS）、PAI＋给药组（水蛭素）大鼠检测面神经核团中PAR-1、Src及p-Src的表达变化。结果 WB及RT-PCR结果显示,SH组各时间点（12、24、72h）PAR-1、Src、p-Src表达水平差异无统计学意义（P〉0.05）;PAI与PAI＋NS组各时间点PAR-1、Src、p-Src表达水平逐渐升高,差异有统计学意义（P〈0.05）;PAI与PAI＋NS组对应时间点比较无统计学意义（P〉0.05）,而PAI＋水蛭素组PAR-1、Src、p-Src表达水平与PAI组相比均显著下降（P〈0.05）。相关分析显示,面神经缺血损伤后PAR-1表达水平与Src磷酸化表达水平呈显著正相关（r=0.884,P〈0.01）。结论大鼠面神经缺血后,PAR-1表达水平不断增加,引起Src及其磷酸化表达水平增加,抑制PAR-1表达后,Src及其磷酸化表达水平下降。说明在面神经损伤和修复中PAR-1对Src及其磷酸化水平有一定的调控作用。     

右美托咪定预处理对局灶性脑缺血/再灌注星形胶质细胞胶质纤维酸性蛋白表达的影响

目的 探讨预注右美托咪定对局灶性脑缺血/再灌注后星形胶质细胞胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)表达的影响。方法 大脑中动脉插线法制作大鼠局灶性脑缺血/再灌注模型。观察脑缺血2 h再灌注24 h后神经功能损害改变并评分,并采用免疫组化和蛋白免疫印迹方法观察大鼠脑缺血后星形胶质细胞GFAP蛋白表达情况。结果 缺血/再灌注后可诱导大鼠脑组织星形胶质细胞GFAP表达明显增强,右美托咪定可明显改善大鼠神经功能损害,减少缺血区GFAP阳性星型胶质细胞,降低GFAP表达水平。结论 早期应用右美托咪定可抑制脑缺血后星形胶质细胞GFAP的过度表达,可发挥抗脑缺血损伤,保护神经元作用。     

原花青素对大鼠局灶性脑缺血再灌注损伤后海马线粒体CytC的释放和Caspase-9的影响研究

目的:研究原花青素对大鼠局灶性脑缺血再灌注所致线粒体损伤的保护作用机制。方法:48只Wistar大鼠随机分为假手术组,缺血再灌注模型组,原花青素高、低剂量(400、40mg.kg-1)组,各组灌胃给予相应试药后30d,采用线栓法阻塞大鼠大脑中动脉(MCA)制作脑缺血再灌注损伤模型,24h后处死取脑。利用免疫荧光方法测定各组小鼠细胞色素C(CytC)的表达,以逆转录聚合酶链反应(RT-PCR)技术检测半胱氨酸蛋白酶(caspase)-9mRNA的表达。结果:与模型组比较,原花青素组CytC阳性细胞明显增多,而caspase-9mRNA表达减弱(P<0.05)。结论:原花青素可以抑制海马CA1区神经元CytC的释放,并减弱cas-pase-9mRNA的表达,对脑缺血再灌注损伤产生保护作用。     

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.     

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.     

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     

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。     

Changes in angiopoietin expression in glomeruli involved in glomerulosclerosis in rats with daunorubicin-induced nephrosis

AIM: To study the potential pathological role of endogenous angiopoietins in daunorubicin-induced progressive glomerulosclerosis in rats. METHODS: Seventy male Wistar rats were allocated randomly into a daunorubicin group (DRB; n=40) or a control group (n=30). The rats in the DRB group were injected with DRB (15 mg/kg), in their tails. Subsequently, at intervals of 1, 2, 4, 6, 8, and 12 weeks, 5 male Wistar rats in each group were chosen randomly for 24 h urinary protein quantitative measurements (24 h UPQM), and determination of plasma tumor necrosis factor alpha (TNF-alpha), angiopoietin-1 (Ang1), and angiopoietin-2 (Ang2) levels. Kidney sections were examined by electron microscopy, Periodic Acid Schiff (PAS) staining, immunohistochemical staining and in situ hybridization histochemistry. RESULTS: As glomerulosclerosis progressed in the DRB group, expression of Ang1 mRNA and protein in glomeruli decreased and expression of TNF-alpha protein, Ang2 mRNA and protein in glomeruli increased. Expression of Ang1 mRNA and protein in glomeruli were negatively correlated with 24 h UPQM, Fn protein expression, and mean area of extracellular matrix (MAECM). In comparison, expression of Ang2 mRNA and protein in glomeruli were positively correlated with 24 h UPQM, Fn protein expression and MAECM; furthermore, there was a positive correlation between plasma Ang2 and 24 h UPQM. Plasma TNF-alpha and expression of TNF-alpha in glomeruli were positively correlated with expression of Ang2 mRNA and protein in glomeruli. There was a negative correlation between Ang1 protein expression and Ang2 protein expression in glomeruli. CONCLUSION: During DRB-induced glomerulosclerosis, podocyte injury led to a shift in the balance of Ang1 and Ang2 in glomeruli. Increased TNF-alpha in plasma and glomeruli may upregulate Ang2 expression in glomeruli. Elevated Ang2 in both plasma and glomeruli may mediate protein permeability through the glomerular filtration barrier. Moreover, local expression of Ang2 may facilitate the progress of glomerulosclerosis by upregulating a component expression of extracellular matrix.     

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.