西洛他唑对糖氧剥离大鼠脑微血管内皮细胞的保护作用

目的探讨磷酸二酯酶(PDE)3抑制剂西洛他唑对大鼠脑微血管内皮细胞糖氧剥离损伤的影响及作用机制。方法原代培养大鼠皮层微血管内皮细胞,建立糖氧剥离细胞模型模拟"缺血"过程,分5组:正常对照组、西洛他唑组、依达拉奉组、溶剂对照组、模型组。糖氧剥离6h后,测定细胞上清中内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS)水平、细胞内环磷腺苷酸(cAMP)浓度,及采用四唑盐(MTT)比色实验测定细胞活力。结果西洛他唑及依达拉奉均可明显提高缺血细胞模型上清中eNOS水平,同时降低iNOS水平,提高细胞内cAMP水平及细胞存活率(均P0.05),且西洛他唑作用更为显著。结论西洛他唑对培养大鼠皮层微血管内皮细胞在缺血损伤中具有保护作用,其作用机制可能是通过选择性抑制PDE3从而增加cAMP水平,间接增加eNOS水平、降低iNOS水平来实现的。     

西洛他唑对大鼠脑微血管内皮细胞缺血再灌注损伤的保护作用

目的研究西洛他唑对大鼠脑微血管内皮细胞缺血再灌注损伤的保护作用机制。方法以原代方法培养大鼠脑微血管内皮细胞并传代,将第3代传代细胞随机分为正常对照组、西洛他唑组、溶剂对照组和缺血再灌注模型组(再灌模型组)4组,对后3组大鼠建立脑微血管内皮细胞糖氧剥夺后复糖氧模型,模拟缺血再灌注过程,并对西洛他唑组和溶剂对照组在造模同时分别以终浓度1×10-5mmol/L西洛他唑〔二甲基亚砜(DMSO)为溶剂〕和0.05%(体积分数)DMSO进行干预。糖氧剥夺3 h复糖氧24 h后,测定各组细胞上清液中诱导型一氧化氮合酶(iNOS)和内皮型一氧化氮合酶(eNOS)水平及细胞内环磷腺苷酸(cAMP)水平,并以四唑盐(MTT)比色实验测定细胞活力。结果西洛他唑组与再灌模型组比较,eNOS水平明显提高,iNOS水平明显降低,cAMP水平及细胞存活率显著提高(均P<0.05)。结论西洛他唑对大鼠脑微血管内皮细胞的缺血再灌注损伤有保护作用,其作用机制可能与促进eNOS水平升高和iNOS水平降低有关。     

西洛他唑对急性脑梗死患者血小板活化功能的作用

目的探讨西洛他唑对急性脑梗死患者血小板活化标志物P-选择素(CD62P)、血小板膜糖蛋白纤维蛋白原受体(PAC-1)的作用。方法 52例急性脑梗死患者,口服西洛他唑胶囊每次100 mg,每日2次;分别于服药前、服药后3 d、1 w和2 w采集外周静脉血,运用流式细胞术检测其CD62P、PAC-1的水平。结果急性脑梗死患者在服用西洛他唑后1 w、2 w外周血CD62P、PAC-1的水平与用药前比较均有明显的下降(P<0.05),用药后3 d CD62P、PAC-1与用药前比较,差异无统计学意义(P>0.05),用药后1 w CD62P、PAC-1和用药后2 w比较,差异无统计学意义(P>0.05)。结论西洛他唑对急性脑梗死患者血小板活化功能具有良好的抑制作用,用药1 w可达到较稳定的状态。     

西洛他唑防治脑卒中的研究进展

西洛他唑(CZ)能够提高细胞内环磷酸腺苷(cAMP)水平,并主要由cAMP介导实现抗血小板聚集等作用.研究发现该药能降低脑卒中风险,减少卒中复发,改善卒中患者预后,其有效性和安全性已得到证实.     

西洛他唑在缺血性卒中二级预防中的应用

磷酸二酯酶（PDE）3西洛他唑是抗血小板聚集药物,具有阻止血栓形成、保护血管内皮功能以及预防动脉粥样硬化进展等作用,减少缺血性卒中事件的发生。本文对近年来西洛他唑的临床应用进展做一综述。     

西洛他唑对全脑缺血大鼠海马磷酸化蛋白激酶B及半胱氨酸天冬酶-3表达的影响

目的研究西洛他唑对全脑缺血大鼠海马磷酸化蛋白激酶B(p-Akt)及半胱氨酸天冬酶-3(caspase-3)的影响。方法用四血管阻断法制作大鼠全脑缺血再灌注模型。将112只(模型成功率为75(,死亡后补充动物)约300g的健康成年SD大鼠随机分为3组:假手术组(n=16)、缺血模型对照组(n=48)及治疗组(n=48)。治疗组于缺血前6h和2h各灌胃给予1次西洛他唑,以后每隔24h在相应时间点给药,其它各组灌胃给予等量二甲基亚砜。缺血模型对照组和治疗组于缺血10min再灌注0h、1h、12h、24h、72h、7d后断头取脑,免疫组织化学方法分析再灌注后不同时间点海马CA1区p-Akt和再灌注后caspase-3表达水平的变化。结果全脑缺血后0h、1h,治疗组海马CA1区p-Akt平均光密度值明显高于模型组(P<0.01;P<0.05)。缺血后24h、72h、7d,治疗组海马CA1区caspase-3平均阳性细胞数明显低于模型组(P<0.05)。结论西洛他唑可以降低大鼠全脑缺血再灌注后海马CA1区caspase-3表达水平,并可抑制再灌注后p-Akt水平的急剧降低。     

西洛他唑和阿司匹林治疗脑梗死后血管性痴呆的临床效果

目的探讨西洛他唑和阿司匹林治疗脑梗死后血管性痴呆的效果。方法我院2010-09—2013-09收治100例脑梗死后血管性痴呆患者。根据患者血压、血糖等情况给予对症处理,观察组给予西洛他唑治疗;对照组给予阿司匹林肠溶片。2组患者采用MMSE量表(简易智能精神状态检查量表)对治疗前后的认知情况进行评定;采用ADL Barthel指数评分法对2组患者治疗前后的日常生活活动能力改变情况进行评定。结果 2组MMSE量表评分、ADL Barthel指数评分治疗前后比较差异有统计学意义(P0.05),观察组间比较差异有统计学意义(P0.05)。观察组治疗后缺血或出血卒中发生率低于对照组,差异有统计学意义(P0.05)。结论西洛他唑在脑梗死后血管性痴呆的治疗中效果显著,在改善患者认知和日常生活活动能力方面优于阿司匹林,值得应用。     

p38MAPK介导神经损伤的研究进展

p38MAPK通路是MAPK信号传导途径中的重要成员。在中枢神经或周围神经损伤后p38MAPK表达发生改变,并且和多种因素相互作用介导神经损伤。本文就p38MAPK信号通路及其在神经损伤中的作用做一综述。     

西洛他唑对大鼠皮层细胞糖氧剥离损伤的保护作用

目的探讨磷酸二酯酶抑制剂西洛他唑对糖氧剥离后大鼠皮层细胞培养的影响及作用机制。方法原代混合培养大鼠皮层细胞,建立糖氧剥离的细胞损伤模型模拟细胞"缺血损伤",然后进行干预。测定细胞培养上清中乳酸脱氢酶(LDH)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、神经元型一氧化氮合酶(nNOS)及诱导型一氧化氮合酶(i NOS)的含量;测定一氧化氮(NO)的分泌水平;测定细胞内环磷酸腺苷(cAMP)水平及四唑盐(MTT)比色试验测定细胞活力。结果西洛他唑组及依达拉奉组与糖氧剥离模型组比较,LDH、MDA漏出量显著减少(P均0.05),GSH-Px释放量明显升高(P均0.05),nNOS、i NOS的水平及NO的分泌量显著下降(P均0.05),细胞内cAMP水平明显升高(P均0.05);细胞存活率显著提高(P均0.05);西洛他唑与依达拉奉组比较,LDH、MDA漏出量及GSH-Px的释放量无差别,nNOS、i NOS和NO的水平明显降低(P均0.05),细胞内cAMP水平显著升高(P0.05);细胞存活率明显提高(P0.05)。结论西洛他唑对培养大鼠皮层细胞在糖氧剥离损伤中具有保护作用,其作用机制可能通过抗氧化、降低nNOS及i NOS的水平从而降低NO的分泌、升高细胞内cAMP水平来实现的。     

西洛他唑调节核因子κB通路抑制炎症因子治疗兔髂动脉成形后的血管内再狭窄

背景：血管成形术后再狭窄发生率高,炎症反应的发生是再狭窄的始动因素,如何有效抑制炎症反应,减少平滑肌细胞迁移和增殖是降低再狭窄发生的关键。多个研究均已证实西洛他唑有确切地降低血管成形术后再狭窄作用,但其作用机制仍不明确。 目的：观察西洛他唑在兔髂动脉粥样硬化球囊成形术后再狭窄中的抗炎作用,揭示其作用途径。 方法：将大白兔分成6组,即空白对照组、血管狭窄组、再狭窄模型组、西洛他唑25,50及100 mg/kg组。高脂饲养,除空白对照组外均制作髂动脉内皮剥脱血管狭窄模型。喂养4周后再狭窄模型组及各西洛他唑治疗组在血管狭窄部位行经皮球囊扩张成形术制作再狭窄模型。血管造影抽血测定血脂浓度及细胞因子质量浓度,以免疫组化法观察核因子κB表达,用图像工作站对血管造影结果行血管狭窄分析。 结果与结论：与再狭窄模型组相比,各西洛他唑治疗组血脂浓度及细胞因子质量浓度均明显下降(P < 0.05)。免疫组化显示再狭窄模型组中核因子κB表达显著高于空白对照组及各西洛他唑治疗组,治疗组随着剂量增加核因子κB表达逐渐减弱(P < 0.05)。血管造影证实,各西洛他唑治疗组与再狭窄模型组相比,血管面积狭窄率及直径狭窄率明显下降(P < 0.01)。结果提示,西洛他唑能够抑制兔髂动脉成形术后再狭窄,通过调节核因子κB抑制各种炎症因子可能是西洛他唑治疗再狭窄的作用途径。     

P38MAPK信号转导通路与血管细胞增殖及缺血再灌注的关系

丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)是广泛表达的丝氨酸/酪氨酸激酶,在真核生物细胞多种信号转导通路中起重要的作用,受多种因素的调控,作用底物多种多样。P38信号通路是MAPK通路中相当重要的一个分支,在炎症、细胞应激、凋亡、细胞周期和生长等多种生理、病理过程中起着重要作用。     

Identification of a mitogen-activated protein kinase site in human myelin basic protein in situ

Ultrastructural localization of a specific phosphorylated isomer of myelin basic protein (MBP) has been achieved with a monoclonal antibody specific for human MBP sequence, 89–105, in which Thr⁹⁸ was phosphorylated. Cryosections of human brain white matter revealed that gold particles were found localized almost exclusively to the major dense line demonstrating that threonine 98 in the sequence Thr-Pro-Arg-Thr-Pro-Pro-Pro, a mitogen-activated protein kinase-specific site, was phosphorylated in vivo. In two cases of multiple sclerosis, the density of gold particles in myelin was reduced by about 30%, in one case by 42%, and by 80% in a fourth case. However, gold labelling was seen in areas of demyelination suggesting that the phosphorylated threonyl peptide was protected from degradation.     

Inhibitors of p38 mitogen-activated protein kinase enhance proliferation of mouse neural stem cells

The p38 mitogen-activated protein kinase (MAPK) is induced in response to environmental stress. Although p38 MAPK has been implicated in diverse cellular processes, including cell proliferation, differentiation, and survival of differentiated cells in the central nervous system (CNS), the expression profile and roles of p38 MAPK in the developing brain remain largely unknown. In the present study, we demonstrate that p38 MAPK is expressed predominantly in nestin-positive cells in the cerebral cortex in embryonic day 10 (E10) brain and that expression of the protein decreases gradually during development. To investigate the roles of p38 MAPK in the embryonic brain, two selective p38 MAPK inhibitors, SB202190 and SB203580, were added to the primary neuronal cultures from E10-E14 brains. After 7 days of exposure to these inhibitors, but not SB202474, a negative analog of SB203580, numerous large neurospheres were present. MAPK inhibitors also selectively increased the growth rate of neural stem cells (NSCs) purified from secondary neurospheres and the number of bromodeoxyuridine-positive NSCs. Thus, p38 MAPK inhibitors are potent stimulators of NSC proliferation, and p38 MAPK may be an intrinsic negative regulator of NSC proliferation during early brain development.     

肝星状细胞增殖与p38丝裂原活化蛋白激酶信号传导通路的关系

背景：肝星状细胞的激活、增殖导致肝纤维化,p38丝裂原活化蛋白激酶信号通路可参与调控细胞增殖。 目的：探讨SB203580作用于乙醛刺激的大鼠肝星状细胞后p38丝裂原活化蛋白激酶活性变化和细胞增殖变化。 方法：体外培养大鼠肝星状细胞株,在乙醛干预的基础上加入不同浓度的p38特异性抑制剂SB203580进行培养,并设置对照。以Western blot检测磷酸化p38 蛋白表达水平变化,MTT比色法检测细胞增殖。 结果与结论：乙醛刺激后大鼠肝星状细胞内磷酸化p38水平增强,细胞增殖明显。使用5,10,20 μmol/L SB203580能明显抑制乙醛刺激的肝星状细胞增殖(P < 0.05),加大浓度至30 μmol/L时,抑制作用更明显(P < 0.01),抑制率为43.9%,而磷酸化p38水平也降低(P < 0.05)。结果证实,抑制p38丝裂原活化蛋白激酶活性可能影响肝星状细胞的增殖。     

Lipopolysaccharide-enhanced transcellular transport of HIV-1 across the blood-brain barrier is mediated by the p38 mitogen-activated protein kinase pathway

Chronic systemic inflammation in the late stage of human immunodeficiency virus type-1 (HIV-1) infection could increase neuroinvasion of infected monocytes and cell-free virus, causing an aggravation of neurological disorders in AIDS patients. We previously showed that the peripheral administration of lipopolysaccharide (LPS) enhanced the uptake across the blood-brain barrier (BBB) of the HIV-1 viral protein gp120. Brain microvessel endothelial cells are targets of LPS. Here, we investigated whether the direct interaction between LPS and the BBB also affected HIV-1 transport using primary mouse brain microvessel endothelial cells (BMECs). LPS produced a dose (1-100 microg/mL)- and time (0.5-4 h)-dependent increase in HIV-1 transport and a decrease in transendothelial electrical resistance (TEER). Whereas indomethacin (cyclooxygenase inhibitor) and L-NAME (NO synthase inhibitor) did not affect the LPS-induced changes in HIV-1 transport or TEER, pentoxifylline (TNF-alpha inhibitor) attenuated the decrease in TEER induced by LPS, but not the LPS-induced increase in HIV-1 transport. LPS also increased the phosphorylation of p44/42 MAPK and p38 MAPK but not that of JNK. U0126 (p44/42 MAPK inhibitor) and SP600125 (JNK inhibitor) did not inhibit the LPS-induced increase in HIV-1 transport although U0126 attenuated the reduction in TEER. SB203580 (p38 MAPK inhibitor) inhibited the LPS-induced increase in HIV-1 transport without affecting TEER. Thus, LPS-enhanced HIV-1 transport is independent of changes in TEER and so is attributed to increased transcellular trafficking of HIV-1 across the BBB. These results show that LPS increases HIV-1 transcellular transport across the BBB by a pathway that is mediated by p38 MAPK phosphorylation in BMECs.     

Involvement of thrombin and mitogen-activated protein kinase pathways in hemorrhagic brain injury

Thrombin is thought to play an important role in brain damage associated with intracerebral hemorrhage (ICH). We previously showed that activation of mitogen-activated protein (MAP) kinases and recruitment of microglia are crucial for thrombin-induced shrinkage of the striatal tissue in vitro and thrombin-induced striatal damage in vivo. Here we investigated whether the same mechanisms are involved in ICH-induced brain injury. A substantial loss of neurons was observed in the center and the peripheral region of hematoma at 3 days after ICH induced by intrastriatal injection of collagenase in adult rats. Intracerebroventricular injection of argatroban or cycloheximide, both of which prevent thrombin cytotoxicity in vitro, exhibited a significant neuroprotective effect against ICH-induced injury. ICH-induced neuron loss was also prevented by a MAP kinase kinase inhibitor (PD98059) and a c-Jun N-terminal kinase inhibitor (SP600125). These drugs had no effect on hematoma size or ICH-induced brain edema. Activation of extracellular signal-regulated kinase in response to ICH was observed in both neurons and microglia. Despite their neuroprotective effects, MAP kinase inhibitors did not decrease the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells appearing after ICH. Identification of cell types revealed that TUNEL staining occurred prominently in neurons but not in microglia, whereas inhibition of MAP kinases resulted in appearance of TUNEL staining in microglia. These results suggest that thrombin and the activation of MAP kinases are involved in ICH-induced neuronal injury, and that neuroprotective effects of MAP kinases are in part mediated by arrestment of microglial activities.     

蛋白激酶C抑制剂Staurosporine与脑膜瘤细胞增生的相互关系

目的研究蛋白激酶C信号传导系统在人体外低代培养脑膜瘤细胞增生中的调节作用。初步探讨蛋白激酶C抑制剂对培养的脑膜瘤细胞增殖的影响及其作用机制。方法选取手术切除的新鲜脑膜瘤组织,常规早期传代细胞培养方法。观察蛋白激酶C抑制剂Staurosporine对细胞增殖的抑制率与基础磷脂酰肌醇(PI)转换率,比较分析上述两者间的相互关系。结果低代培养脑膜瘤细胞的增殖对蛋白激酶C抑制剂Staurosporine表现出剂量依赖性抑制效应。蛋白激酶C抑制剂对细胞增殖的抑制率与基础PI转换率之间存在正相关,r=0.58,P<0.01。结论蛋白激酶C信号传导系统介入人体外低代培养脑膜瘤细胞增生的调节。     

VEGF及PCNA表达与脑胶质瘤分级的相关性研究

目的　研究血管内皮细胞生长因子 (VEGF)与增殖性细胞核抗原 (PCNA)蛋白在各级脑胶质瘤中的表达率及与胶质瘤病理分级的关系。方法　利用免疫组化方法检测 4 5例胶质瘤手术标本中VEGF与PCNA蛋白的表达水平 ,并比较二者的相关性。结果　随着胶质瘤病理级别的升高 ,VEGF及PCNA的表达率渐之升高 ,Ⅰ Ⅳ级病理分组中 ,VEGF阳性率分别为 2 2 .2 9± 7.6 4 ,5 3.75± 10 .74 ,70 .2 7± 14 .2 7,PCNA阳性率分别为 15 .86± 4 .5 5 ,32 .4 1± 7.4 4 ,4 7.92± 10 .31,且并各级之间有显著差异 (P <0 .0 5 )。结论　VEGF与PCNA蛋白的表达对判断胶质瘤病理分级及生物学行为有一定意义。     

三氧化二砷对大鼠血管平滑肌细胞增殖与凋亡的影响

目的观察三氧化二砷(As2O3)对大鼠血管平滑肌细胞(VSMC)增殖及凋亡的影响,以提供含As2O3缓释血管内支架植入手术后防止再狭窄的实验依据。方法分离培养大鼠VSMC并以As2O3处理;用四甲基偶氮唑蓝还原反应(MTT法)检测细胞增殖情况,RT-PCR检测增殖细胞核抗原(PCNA)mRNA;以AnnexinV+PI双染色流式细胞术检测细胞凋亡;比色法检测半胱氨酸天门冬氨酸蛋白酶-3(Caspase-3)的活性。结果 2μmol/L的As2O3对VSMC的生长有明显的抑制作用,且此作用呈时间依赖性(P<0.01)。以2μmol/L的As2O3作用72h后,AS2O3对PCNAmRNA表达有显著的抑制作用(P=0.009),对VSMC凋亡的发生(P=0.0001)及对Caspase-3的激活(P=0.005)均有显著促进作用。结论 As2O3可有效抑制VSMC增殖,诱导VSMC凋亡。As2O3的应用有可能为防治血管内支架植入术后再狭窄的药物研究提供一个新思路。     

A new factor derived from 1321N1 human astrocytoma cells causes differentiation of PC-12 cells mediated through mitogen-activated protein kinase cascade

Glial cells play an important role in maintaining neural function. In the present study, we examined the effects of a factor derived from human astrocytoma cells (1321N1) on differentiation of rat pheochromocytoma cells (PC-12). The conditioned medium which had been used for culture of 1321N1 cells caused the differentiation of PC-12 cells, suggesting that 1321N1 cells release a neurotrophic factor. The factor was apparently distinct from well-known neurotrophic factors, such as nerve growth factor (NGF), since it was resistant to boiling and trypsin treatment. The molecular size of the factor was assumed to be below 1000 through dialysis and ultrafiltration experiments. Furthermore, PC-12 cells were differentiated synergistically by the combined addition of NGF and the conditioned medium of 1321N1 cells. Partially purified fraction of the factor by Sephadex G-15 gel filtration column caused the prolonged activation of mitogen-activated protein kinase (MAPK). The differentiation of PC-12 cells induced by the fraction or NGF disappeared after the treatment with PD98059, a specific inhibitor of MAPK kinase (MEK), suggesting the involvement of MAPK in the differentiation. These results suggest that the new low-molecular factor derived from glial cells causes differentiation of PC-12 cells mediated through an activation of MAPK.