HCV核心蛋白抑制干扰素α诱导的抗病毒基因表达及其机制研究

研究HCV核心蛋白对干扰素α诱导的抗病毒分子PKR和2′-5′OAS表达的影响及其机制。HCV核心蛋白表达质粒转染HepG2细胞,RT-PCR分析PKR和2′-5′OAS的mRNA水平变化,荧光素酶活性分析核心蛋白对ISRE介导的基因表达的影响;Western-blot分析SOCS3、STAT1及STAT1磷酸化水平的变化。在干扰素α刺激情况下,表达HCV核心蛋白的细胞中,PKR和2′-5′OAS的mRNA水平下降,ISRE介导的荧光素酶活性降低,STAT1磷酸化水平下降。此外,核心蛋白表达的细胞中SOCS3的mRNA和蛋白水平明显升高。结果表明,HCV核心蛋白可能通过激活SOCS3、抑制STAT1的磷酸化,从而下调干扰素α诱导的PKR和2′-5′OAS表达。     

HCV核心蛋白抑制干扰素α诱导的抗病毒基因表达及其机制研究

研究HCV核心蛋白对干扰素α诱导的抗病毒分子PKR和2′-5′OAS表达的影响及其机制。HCV核心蛋白表达质粒转染HepG2细胞,RT-PCR分析PKR和2′-5′OAS的mRNA水平变化,荧光素酶活性分析核心蛋白对ISRE介导的基因表达的影响;Western-blot分析SOCS3、STAT1及STAT1磷酸化水平的变化。在干扰素α刺激情况下,表达HCV核心蛋白的细胞中,PKR和2′-5′OAS的mRNA水平下降,ISRE介导的荧光素酶活性降低,STAT1磷酸化水平下降。此外,核心蛋白表达的细胞中SOCS3的mRNA和蛋白水平明显升高。结果表明,HCV核心蛋白可能通过激活SOCS3、抑制STAT1的磷酸化,从而下调干扰素α诱导的PKR和2′-5′OAS表达。     

JAK-STAT参与血管平滑肌细胞血管紧张素原和心钠素基因的转录激活

为探讨Janus蛋白酪氨酸激酶2-转导及转录激活因子5（JAK2-STAT5）途径在介导血管紧张素Ⅱ（AngⅡ）诱导的血管平滑肌细胞（VSMC）血管舒-缩肽表达调节的作用及分子机制, 以Ang Ⅱ为诱发因素刺激培养的大鼠VSMC, 用免疫共沉淀、Western印迹分析和激光共聚焦显微镜观察STAT5磷酸化及其核转位, 用电泳迁移率改变分析（EMSA）确定STAT5与血管活性肽基因调控区顺式调控元件的结合活性. 结果显示, STAT5磷酸化水平分别于Ang Ⅱ刺激10 min和12 h出现两个高峰, 增加的磷酸化STAT5主要分布在细胞核内. Ang Ⅱ诱导的STAT5活化与核转位可被JAK2的特异抑制剂AG490所抑制. EMSA结果显示, 用Ang Ⅱ刺激VSMC后, 核蛋白与含有血管紧张素原基因启动子STAT5识别序列的探针结合活性显著升高,而核蛋白与含有心钠素（ANF）基因启动子STAT5识别序列的探针结合活性则呈下降趋势, 核蛋白与两种探针的结合活性均可被JAK2抑制剂AG490所消除, 并且加入抗STAT5抗体后均可出现滞后的超迁移带. 结果提示, Ang Ⅱ通过激活JAK2-STAT5介导信号向胞核内传递, STAT5与相应的顺式元件结合是启动血管紧张素原和心钠素基因表达所必需的转录调控机制之一.     

肺腺癌细胞中STAT3表达对细胞生长及药物敏感性的影响

目的:探讨STAT3表达变化对细胞生长及化疗药物敏感性的影响.方法:采用AG490处理细胞、SOCS3基因转染A549细胞后.Western blot检测STAT3蛋白酪氨酸磷酸化水平变化;MTT法检测细胞增殖情况;不同浓度泰素处理细胞后观察细胞对药物的敏感性.结果:AG490处理细胞、SOCS3基因转染细胞后,Western blot证实其能显著抑制STAT3蛋白酪氨酸磷酸化水平(P<0.01);MTT法结果示细胞增殖明显受到抑制;细胞对泰素敏感性显著增高.结论:STAT3能促进细胞增殖,AG490、SOCS3能显著抑制A549细胞中STAT3蛋白的活性,从而抑制A549细胞生长并增加其对化疗药物的敏感性.     

genistein对沙眼衣原体感染细胞信号转导的影响

研究酪氨酸激酶抑制剂 genistein对IFN γ诱导沙眼衣原体感染细胞内信号转导的影响。以IFN γ作用于沙眼衣原体 (K血清型 )感染的McCoy细胞 ,用 genistein阻断IFN γ对沙眼衣原体感染细胞的作用。用台盼蓝染色法检测沙眼衣原体感染细胞存活率。用免疫印迹法检测蛋白酪氨酸激酶磷酸化及JAK1/STAT1活化的改变。结果显示 :genistein可拮抗IFN γ降低沙眼衣原体感染细胞存活率的作用 ,且具有剂量依赖效应。genistein可抑制IFN γ诱导的沙眼衣原体感染细胞蛋白酪氨酸激酶磷酸化及JAK1/STAT1活化 ,此作用且与genistein作用的剂量有关。     

酪氨酸磷酸化蛋白在体外获能豚鼠精子上的分布与表达

为研究豚鼠精子获能过程中蛋白酪氨酸磷酸化的变化规律,将豚鼠精子悬浮于改良的TALP获能培养基中,在5% CO2 孵箱37 ℃培养,以精子与金霉素(CTC) 荧光结合类型为指标评价精子获能状态,用免疫荧光技术和Western blot方法检测酪氨酸磷酸化的蛋白在精子上的分布以及酪氨酸磷酸化水平的变化。结果显示,随着获能的进行,发生蛋白酪氨酸磷酸化的精子占总精子的百分比增加,由未获能前的36％增至获能7h时的92％。酪氨酸磷酸化的蛋白分布变广,由精子头部扩增至精子头部、鞭毛主段和中段。另外,有80,45,40kDa的三种蛋白发生酪氨酸磷酸化,其中40kDa的蛋白酪氨酸磷酸化水平自精子体外培养后呈递增趋势,45kDa的蛋白酪氨酸磷酸化自培养3h后发现并呈递增趋势,而80kDa的蛋白酪氨酸磷酸化水平在精子培养3h时最高,后呈递减趋势。     

脑缺血Akt 和MAPK 磷酸酶负性调节c-Jun N 端激酶信号通路

目的:探讨脑缺血再灌后Akt和MAPK磷酸酶与JNK活性下调的关系。方法:采用成年清洁级雄性SD大鼠,建立四动脉阻断前脑缺血再灌注模型。缺血10min后再灌注不同时间(15min,1h,4h,24h)。侧脑室分别给予PI3K抑制剂LY294002(LY)和MAPK磷酸酶抑制剂放线菌酮(CHO)。免疫印迹观察p-Akt和p-JNK蛋白水平变化。结果:脑缺血再灌注4h,JNK的活性能被Akt抑制剂LY294002增强,表明激活的Akt能够下调JNK信号通路。而MAPK磷酸酶抑制剂放线菌酮能上调缺血后JNK活性,提示MAPK磷酸酶通过去磷酸化参与了JNK的活性抑制。结论:前脑缺血再灌后,激活Akt和MAPK磷酸酶参与了JNK信号通路负性调节,是抑制JNK诱导缺血后中枢神经损伤的重要机制。     

血管内皮生长因子受体1酪氨酸激酶的克隆、表达和功能

血管内皮生长因子受体 1(Flt 1)在血管生成过程中起着重要的作用。Flt 1胞内域的酪氨酸激酶直接参与了VEGF与Flt 1结合后的胞内信号转导途径。在原核系统中表达得到具有酶活性的Flt 1酪氨酸激酶融合蛋白 ,并进行了初步的性质研究。利用逆转录PCR技术从人肝癌组织总RNA中得到Flt 1酪氨酸激酶区的cDNA ,将其克隆到表达载体质粒 pGEX KG中 ,并在大肠杆菌BL2 1(DE3) pLysS中表达、纯化 ,得到可溶的Flt 1酪氨酸激酶融合蛋白 (GST F)。虽然GST F不包含目前已知的磷酸化位点 ,但研究表明GST F能够进行自磷酸化反应 ,并且其活性需要镁离子或锰离子的参与。同时发现GST F能够磷酸化合成底物 polyE4Y ,而不能作用于MBP和Src相关肽。底物磷酸化时最适的镁离子和锰离子浓度分别为 15mmol/L和 0 .5mmol/L。GST F为寻找抗肿瘤药物提供了一个有效工具     

沙土鼠脑缺血和再灌流后钙调素蛋白激酶Ⅱ活性变化及其与脑电图的关系

结扎蒙古沙土鼠双侧颈总动脉制作急性脑缺血模型,观察了急性脑缺血和再灌流后钙调素依赖性蛋白激酶Ⅱ(CaM-PKⅡ)和蛋白磷酸酶(CaM-PrP)活性的变化以及脑电图(EEG)的表现。结果表明:(1)CaM-PKⅡ活性随缺血时间延长而逐渐降低,缺血10min酶活性即显著降低;(2)缺血10min再灌流,CaM～PKⅡ活性可部分恢复;(3)急性脑缺血时CaM-PrP活性无明显改变;(4)缺血10min再灌流,EEG在1h基本恢复正常,缺血20min再灌流,EEG在3h仍未恢复正常。上述结果提示:CaM-PKⅡ活性对缺血非常敏感,而CaM-PrP活性对缺血不敏感,脑缺血再灌流后EEG的表现与CaM-PKⅡ活性有一定的相关性。     

蛋白酪氨酸磷酸酶SHP-2与疾病的相关性研究进展

蛋白酪氨酸磷酸酶家族由130多种蛋白酪氨酸磷酸酶组成,它们和蛋白质酪氨酸激酶家族一起调控蛋白质中酪氨酸残基的磷酸化以及去磷酸化的动态平衡,它们的活性直接决定细胞内蛋白质的磷酸化水平的高低。SHP-2是蛋白酪氨酸磷酸酶家族的一员,在各种细胞和组织中均有广泛的表达,参与多个信号传导通路,介导细胞的生长、分化、迁移、粘附及凋亡等。SHP-2的表达异常会导致多种疾病的产生,但是相关综述较少,同时未见文献报道其在胶质瘤中的作用,因此本文简要介绍SHP-2的结构、功能、信号传导,并阐述了SHP-2与常见疾病的关系。     

脑缺血和缺血再灌注大鼠皮层神经元自噬的动态变化

目的：探讨脑缺血和缺血/再灌注不同时间大鼠大脑皮层神经元自噬的变化。方法：健康雄性SD大鼠60只,随机分为：假手术（Sham）组（n=10）,脑缺血和缺血/再灌注模型组（n=50）.模型组分别在缺血30min、2h,缺血2h再灌注1h、6h、24h五个时间点,随机抽取10只大鼠,测定脑梗死体积和脑含水量,同时采用Western印迹法测定各组大鼠大脑皮层中微管相关蛋白轻链3-Ⅱ（LC3-Ⅱ）的水平,透射电镜检测大脑皮层神经细胞自噬情况。结果：脑缺血30min时LC3-Ⅱ/Ⅰ比值未见明显上升,缺血2h时LC3-Ⅱ/Ⅰ比值开始升高,明显高于Sham组（P<0.01）;缺血/再灌注1h、6h时LC3-Ⅱ/Ⅰ比值虽较缺血2h组有所下降,但仍明显高于Sham组（P<0.05）;缺血/再灌注24h时LC3Ⅱ/Ⅰ比值达高峰,明显高于Sham组（P<0.01）。透射电镜观察进一步证实该现象。缺血/再灌注6h和24h时大鼠脑梗死体积明显增加,与Sham组比较有统计学差异（P<0.01）。缺血/再灌注24h大鼠脑组织含水量明显增加,明显高于Sham组（P<0.05）。HE染色显示：仅在缺血/再灌注24h组大鼠皮层见组织水肿、疏松,部分细胞变性、凋亡,海马区见大量神经元细胞核皱缩、深染呈变性凋亡状。结论：局灶性脑缺血和缺血/再灌注模型中大脑皮层缺血2 h神经元自噬即明显激活,缺血/再灌注1 h、6 h自噬均持续增高,缺血/再灌注24 h自噬达高峰。     

Activated mitogen-activated protein kinase kinase 7 redistributes to the cytosol and binds to Jun N-terminal kinase-interacting protein 1 involving oxidative stress during early reperfusion in rat hippocampal CA1 region

Mitogen-activated protein kinase kinase (MKK) 7, a specific upstream activator of Jun N-terminal kinases (JNKs) in the stress-activated protein kinase (SAPK)/JNK signaling pathway, plays an important role in response to global cerebral ischemia. We investigated the subcellular localization of activated (phosphorylated) MKK (p-MKK) 7 using western blotting, immunoprecipitation and immunohistochemistry analysis in rat hippocampus. Transient forebrain ischemia was induced by the four-vessel occlusion method on Sprague-Dawley rats. Our results showed that both protein expression and activation of MKK7 were increased rapidly with peaks at 10 min of reperfusion in the nucleus of the hippocampal CA1 region. Simultaneously, in the cytosol activated MKK7 enhanced gradually and peaked at 30 min of reperfusion. In addition, we also detected JNK-interacting protein (JIP) 1, which accumulated in the perinuclear region of neurons at 30 min of reperfusion. Interestingly, at the same time-point the binding of JIP-1 to p-MKK7 reached a maximum. Consequently, we concluded that MKK7 was rapidly activated and then translocated from the nucleus to the cytosol depending on its activation in the hippocampal CA1 region. To further elucidate the possible mechanism of MKK7 activation and translocation, the antioxidant N-acetylcysteine was injected into the rats 20 min before ischemia. The result showed that the levels of MKK7 activation, translocation and binding of p-MKK7 to JIP-1 were obviously limited by N-acetylcysteine in the cytosol at 30 min after reperfusion. The findings suggested that MKK7 activation, translocation and binding to JIP-1 were closely associated with reactive oxygen species and might play a pivotal role in the activation of the JNK signaling pathway in brain ischemic injury.     

脑预缺血引起大鼠海马细胞膜腺苷受体数量和亲和力升高及其对神经元的保护作用

采用放射性配基结合法,测定大鼠全脑缺血后海马细胞膜腺苷(adenosine,ADO)受体数量及亲和力的变化,以探讨其与脑缺血耐受形成之间的关系。发现缺血6min即可导致海马组织明显的神经元延迟性死亡(delayed neuron     

15-HETE参与的脑缺血再灌注损伤中p-ERK1/2表达变化的研究

目的:通过应用15-脂氧化酶(15-Lipoxygenase,15-LOX)抑制剂去甲二氢愈创木酸(nordihydroguaiaretic acid,NDGA)抑制15-羟基-二十碳四烯酸(15-hydroxyeicosatetraenoic acid,15-HETE)的生成,观察缺血再灌注损伤中大鼠脑组织磷酸化细胞外信号调节酶(phosphor-extracellular signal-regulated kinase,p-ERK1/2)表达的变化,探讨p-ERK1/2在15-HETE参与的脑缺血再灌注损伤中的作用及其表达变化。方法:应用大脑中动脉线栓栓塞法(MCAO)制作大鼠脑梗死2小时再灌注模型。将大鼠随机分为三组:假手术组(sham组)、DMSO对照组、NDGA处理组。后两组再根据不同的灌注时间分为三个亚组:再灌注1小时组、再灌注6小时组、再灌注24小时组。采用TTC染色法检测再灌注24小时大鼠脑梗死体积;免疫印迹(Western blot)法测定梗死后再灌注不同时间点梗死核心区和梗死周围区的p-ERK1/2的表达。结果:假手术组仅有少量p-ERK1/2的表达。DMSO对照组梗死核心区p-ERK1/2的表达从梗死再灌注后1小时即开始逐渐升高(1.43±0.06),6小时达高峰(2.02±0.14),24小时有所下降(1.16±0.21),与假手术组(0.62±0.08)比较P值均0.01;梗死周围区p-ERK1/2表现出相同的变化趋势。与DMSO对照组比较,NDGA处理组大鼠脑梗死体积显著减小(20.10±0.12%vs 17.24±0.16%,P=0.009,P0.05),各时间点p-ERK1/2的表达均下降。与梗死核心区相比较,梗死周围区24小时仍可检测到较高含量的p-ERK1/2(1.16±0.21 vs 1.86±0.14),但梗死核心区表达相对较少。结论:脑缺血再灌注损伤中,p-ERK1/2的表达增加,说明p-ERK1/2参与其中;应用NDGA后,p-ERK1/2的表达降低,脑梗死体积减小,证实p-ERK1/2参与了15-HETE介导的脑缺血再灌注损伤,并在此过程中可能参与了细胞的凋亡。     

20-羟基蜕皮甾酮对大鼠全脑缺血再灌注后海马神经元损伤和炎症反应的影响

目的:观察20-羟基蜕皮甾酮对全脑缺血再灌注后SD大鼠海马神经元和认知功能的保护作用,并探讨其相关机制。方法:采用四血管闭塞法建立SD大鼠全脑缺血再灌注模型,脑电图和脑组织Nissl染色评估模型的可靠性。将实验动物分为假手术组,缺血再灌注组和缺血再灌注+20-羟基蜕皮甾酮组。TUNEL染色观察海马神经元凋亡,Morris水迷宫实验评价大鼠的认知功能,酶联免疫法测定缺血再灌注后3-24小时大鼠血清中白细胞介素-1β(IL-1β)和肿瘤坏死因子α(TNF-α)的浓度。结果:全脑缺血再灌注后大鼠海马神经元凋亡率从4.50±1.90%上升至72.90±8.40%(p0.01),给予20和40 mg/kg 20-羟基蜕皮甾酮干预,大鼠海马神经元凋亡率分别下降至51.40±8.60%(p0.05)和42.70±6.80%(p0.01)。与假手术组相比,全脑缺血再灌注后大鼠在Morris水迷宫定位航行试验中逃避潜伏期明显延长(p0.01),在空间探索试验中目标象限停留时间和穿越目标象限次数明显减少(p0.01),而20-羟基蜕皮甾酮显著抑制上述变化,改善大鼠的认知功能。缺血再灌注后3-24小时,大鼠血清中IL-1β和TNFα浓度较假手术组显著升高,20-羟基蜕皮甾酮能抑制上述各时间点大鼠血清中IL-1β和TNFα浓度的升高。结论:20-羟基蜕皮甾酮对全脑缺血再灌注后大鼠海马神经元和认知功能有显著保护作用,抑制缺血再灌注后的炎症反应是其保护机制之一。     

Reversibility of Nimodipine Binding to Brain in Transient Cerebral Ischemia

Using autoradiography, we have measured the in vivo binding of [3H]nimodipine to brain in a rat model of reversible cerebral ischemia. Ischemia was induced by simultaneous occlusion of the middle cerebral artery (MCA) and ipsilateral common carotid artery by microaneurysm clips. Rats were studied after 15 min of ischemia (ischemic group) or after 45 min of reperfusion following 15 min of ischemia (reperfused group). Regional cerebral blood flow (CBF) was determined autoradiographically using [14C]iodoantipyrine in both ischemic (n = 6) and reperfused (n = 6) groups. During ischemia blood flow in the territory of the MCA was depressed and recovered to normal only in the distal territory of the MCA following reperfusion. [3H]Nimodipine binding in the ischemic group (n = 12) was elevated in ischemic brain regions and declined significantly (p < 0.01) in these regions in the reperfused group (n = 11). The ratio of the volume of cortex showing increased binding to the total volume of the forebrain was 0.113 +/- 0.025 (mean +/- SD) in the ischemic group and declined to 0.080 +/- 0.027 following reperfusion (p < 0.005). In general, infarct was only observed in regions showing persistent elevation of nimodipine binding following reperfusion as determined by histology performed in a separate group of rats (n = 8) after 24 h of reperfusion. We conclude that increased nimodipine binding to ischemic tissue is initially reversible with prompt reestablishment of CBF and is a sensitive indicator of early and reversible ischemia-induced cerebral dysfunction.     

Autophagy Activation Contributes to the Neuroprotection of Remote Ischemic Perconditioning Against Focal Cerebral Ischemia in Rats

Remote ischemic perconditioning (RIPer) has been proved to provide potent cardioprotection. However, there are few studies on neuroprotection of RIPer. This study aims to clarify the neuroprotective effect of RIPer and the role of autophagy induced by RIPer against cerebral ischemia reperfusion injury in rats. Using a transient middle cerebral artery occlusion (MCAO) model in rats to imitate focal cerebral ischemia. RIPer was carried out 4 cycles of 10 min ischemia and 10 min reperfusion, with a thin elastic band tourniquet encircled on the bilateral femoral arteries at the start of 10 min after MCAO. Autophagy inhibitor 3-methyladenine (3-MA) and autophagy inducer rapamycin were administered respectively to determine the contribution of autophagy in RIPer. Neurologic deficit scores, infarct volume, brain edema, Nissl staining, TUNEL assay, immunohistochemistry and western blot was performed to analyze the neuroprotection of RIPer and the contribution of autophagy in RIPer. RIPer significantly exerted neuroprotective effects against cerebral ischemia reperfusion injury in rats, and the autophagy-lysosome pathway was activated by RIPer treatment. 3-MA reversed the neuroprotective effects induced by RIPer, whereas rapamycin ameliorated the brain ischemic injury. Autophagy activation contributes to the neuroprotection by RIPer against focal cerebral ischemia in rats.     

STAT-3 activation is necessary for ischemic preconditioning in hypertrophied myocardium

The JAK-STAT pathway is activated in the early and late phases of ischemic preconditioning (IPC) in normal myocardium. The role of this pathway and the efficacy of IPC in hypertrophied hearts remain largely unknown. We hypothesized that phosphorylated STAT-3 (pSTAT-3) is necessary for effective IPC in pressure-overload hypertrophy. Male Sprague-Dawley rats 8 wk after thoracic aortic constriction (TAC) or sham operation underwent echocardiography and Langendorff perfusion. Randomized hearts were subjected to 30 min of global ischemia and 120 min of reperfusion with or without IPC in the presence or absence of the JAK-2 inhibitor AG-490 (AG). Functional recovery and STAT activation were assessed. TAC rats had a 31% increase in left ventricular mass (1,347 +/- 58 vs. 1,028 +/- 43 mg, TAC vs. sham, P < 0.001), increased anterior and posterior wall thickness but no difference in ejection fraction compared with sham-operated rats. In TAC, IPC improved end-reperfusion maximum first derivative of developed pressure (+dP/dt(max); 4,648 +/- 309 vs. 2,737 +/- 343 mmHg/s, IPC vs. non-IPC, P < 0.05) and minimum -dP/dt (-dP/dt(min); -2,239 +/- 205 vs. -1,215 +/- 149 mmHg/s, IPC vs. non-IPC, P < 0.05). IPC increased nuclear pSTAT-1 and pSTAT-3 in sham-operated rats but only pSTAT-3 in TAC. AG in TAC significantly attenuated +dP/dt(max) (4,648 +/- 309 vs. 3,241 +/- 420 mmHg/s, IPC vs. IPC + AG, P < 0.05) and -dP/dt(min) (-2,239 +/- 205 vs. -1,323 +/- 85 mmHg/s, IPC vs. IPC + AG, P < 0.05) and decreased only nuclear pSTAT-3. In myocardial hypertrophy, JAK-STAT signaling is important in IPC and exhibits a pattern of STAT activation distinct from nonhypertrophied myocardium. Limiting STAT-3 activation attenuates the efficacy of IPC in hypertrophy.     

Changes of Endogenous Antioxidant Enzymes during Ischemic Tolerance Acquisition

The purpose of this study was to investigate the role of superoxide dismutase (SOD) and catalase (CAT) in brain ischemic tolerance induced by ischemic preconditioning. Forebrain cerebral ischemia was induced in rat by four vessel occlusion. The activities of the antioxidant enzymes CuZn-SOD, Mn-SOD and CAT were measured in the hippocampus, striatum and cortex after 5 min of ischemia used as a preconditioning and subsequent reperfusion, by spectrophotometric methods. In all ischemia-reperfusion groups (5 h, 1 and 2 days of reperfusion), CuZn-SOD activities were found to be increased if compared to the sham operated controls. The increase was significant (P < 0.05) in all reperfusion groups, particularly after 5 h of reperfusion (3 times) in all studied brain regions; the largest increase was detected in the more vulnerable hippocampus and striatum. Very similar changes were found in Mn-SOD activity. The activity of CAT was increased too, but reached the peak of postischemic activity 24 h after ischemia. Our attempt to understand the mechanisms of increased SOD and CAT activities by application of protein synthesis inhibitor cycloheximide showed that this increase was caused by de novo synthesis of enzymes during first hours after ischemia. Our findings indicate that both major endogenous antioxidant enzymes SOD and CAT are synthesized as soon as 5 h after ischemia. In spite of significant upregulation of these enzymes a large number of neurons in selectively vulnerable CA1 region of hippocampus undergoes to neurodegeneration within 7 days after ischemia.     

Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia

We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT)-positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.