东莨菪碱致记忆障碍大鼠中枢电压依赖性钾通道亚型的表达

目的研究东莨菪碱致记忆障碍大鼠模型中枢电压依赖性钾通道亚型mRNA表达的差异。方法Morris水迷宫实验检验大鼠空间学习记忆能力,用RT-PCR方法检测大脑皮层和海马中5种电压依赖性钾通道Kv1.4,Kv1.5,Kv2.1,Kv4.2及Kv4.3 mRNA的表达。结果注射东莨菪碱大鼠的学习记忆能力明显下降,大脑皮层中Kv4.2的表达比对照组降低28.8%;海马中Kv1.4的表达升高111.7%,Kv2.1的表达升高64.3%,而Kv4.2的表达降低33.9%。其它电压依赖性钾通道表达的变化不大。结论东莨菪碱致大鼠学习记忆障碍的同时可诱发中枢电压依赖性钾通道亚型的表达改变。     

慢性孵育β淀粉样肽25-35对培养大鼠海马神经元电压依赖性外向钾通道亚型mRNA表达的影响

目的研究慢性孵育β淀粉样肽_25-35(β-AP_25-35)对海马神经元电压依赖性外向钾通道亚型mRNA表达的影响。方法用RT-PCR方法检测mRNA的表达,用光密度扫描法半定量测定表达变化。结果在正常培养的海马神经元上延迟整流(Kv2.1,Kv1.5),瞬间外向(A型)(Kv4.2,Kv1.4),钙激活的大电导(rSlo)钾通道亚型均有表达。β-AP_25-35 3 μmol·L^-1孵育细胞24 h后,Kv2.1 mRNA的表达明显上调,其它亚型则无显著性变化;β-AP_25-35上调Kv2.1 mRNA的作用主要发生在β-AP_25-35应用后48 h内;60 h后Kv2.1 mRNA表达水平显著下调。结论Kv2.1转录水平的上调可能参与β-AP_25-35选择性地增加海马神经元上延迟整流钾电流(I_K)的作用。     

Kv通道亚型在15-HETE收缩肺动脉过程中的作用

目的从组织功能及细胞分子水平研究电压门控型钾通道(Kv通道)亚型在15-羟化二十碳四烯酸(15-HETE)致大鼠肺动脉收缩过程中的作用。方法采用组织浴槽血管环法，使用Kv通道阻断剂，确定受15-HETE调控大鼠肺动脉平滑肌细胞(PASMCs)膜上Kv亚型；使用RT-PCR和Western blotting技术观察受15-HETE调控PASMCs 膜上Kv亚型。结果阻断Kv1.1，Kv1.2，Kv1.3和Kv1.6通道并不影响15-HETE诱导肺动脉血管收缩；15-HETE不影响PASMCs膜上Kv1.1和Kv1.2通道蛋白质表达；15-HETE下调PASMCs膜上Kv1.5和Kv2.1通道mRNA和蛋白质表达。结论缺氧可能是通过15-HETE这一介导因子抑制Kv1.5和Kv2.1通道，减少PASMCs膜上功能性Kv1.5和Kv2.1通道数量，导致PASMCs收缩。     

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。     

曲美他嗪对2型糖尿病模型大鼠心室肌Ito通道表达的影响研究

目的:探讨曲美他嗪(TMZ)对2型糖尿病模型大鼠心室肌Ito通道α亚单位(Kv1.4、Kv4.2、Kv4.3)表达的影响。方法:大鼠高脂喂养1月后,一次性腹腔注射链脲佐菌素35mg·kg-1建立2型糖尿病模型,建模成功后,随机分为治疗组(高脂饮食,灌胃TMZ10mg·kg-1)和模型组(高脂饮食,灌胃生理盐水),每组10只,给予相应药物,每天1次,4周后分别用实时荧光定量聚合酶链式反应技术(Real-timePCR)、蛋白免疫印迹法(Westernblot)检测左心室Kv1.4、Kv4.2、Kv4.3mRNA和蛋白质的表达水平,另设正常对照组进行比较。结果:与正常对照组比较,模型组大鼠左心室Kv1.4mRNA和蛋白质表达明显升高,Kv4.2、Kv4.3mRNA和蛋白质表达明显降低(P均<0.01);与模型组比较,治疗组大鼠左心室Kv1.4mRNA和蛋白质表达明显降低,Kv4.2、Kv4.3mRNA和蛋白质表达明显升高(P均<0.05)。结论:TMZ可逆转2型糖尿病模型大鼠心室肌Ito通道α亚单位表达的变化。     

小檗碱对局灶性脑缺血大鼠原癌基因c—fos表达的影响

目的：研究大鼠局灶性脑缺血过程中c-fos mRNA表达的动态变化，并观察小檗碱（Ber）对其作用。方法：运用斑点杂交技术，观察Ber对局灶性脑缺血再灌注大鼠大脑皮质及海马组织c-fos mRNA表达的影响。结果：大鼠局灶性脑缺血2h再灌注0．5－4h，脑皮质及海马组织c-fos mRNA出现一过性高表达，2h达高峰，分别为对照组的4．5和4．7倍。Ber 20mg/(kg·d)ip显抑制大鼠脑缺血诱导的c-fos高表达，降低病灶侧海马和皮层组织水、钙含量。结论：脑缺血过程中c-fos原癌基因呈现一过性高表达，Ber可降低c-fos mRNA水平，该作用可能是其抗脑缺血机理之一。     

β-淀粉样肽25-35致记忆障碍大鼠中枢双孔钾通道亚型mRNA表达的改变

目的研究聚集态β-淀粉样肽_25-35（β-AP_25-35）致记忆障碍大鼠模型中枢双孔钾通道亚型mRNA表达的变化。方法Morris水迷宫实验检验大鼠空间学习记忆能力，用RT-PCR方法检测大脑皮层和海马中3种双孔钾通道TREK-1,TREK-2和TRAAK mRNA的表达。结果在Morris水迷宫实验中，β-AP_25-35注射组大鼠d 1，d 2和d 4的潜伏期均比对照组显著延长。β-AP_25-35注射组大鼠大脑海马中TREK-1,TREK-2和TRAAK mRNA的表达比对照组明显升高；而在皮层中的表达变化不显著。结论侧脑室注射聚集态β-AP_25-35致大鼠学习记忆障碍的同时可诱发中枢双孔钾通道亚型的表达改变。     

丙泊酚对大鼠脑缺血再灌注损伤的保护作用及其Caspase-3表达干预机制的研究

目的通过观察ip丙泊酚对脑缺血再灌注大鼠海马区神经细胞的影响,探讨丙泊酚对临床围手术期预防脑缺血再灌注损伤的作用。方法 SD大鼠60只随机分为丙泊酚组、假手术组和模型组。采用大脑中动脉线栓法制作左侧大脑动脉栓塞模型。在缺血再灌注前10 min时,丙泊酚组ip丙泊酚50 mg/kg,模型组ip给予等量生理盐水。假手术组只进行颈部切开、缝合操作而不进行缺血再灌注实验。缺血再灌注24 h采集脑组织。TTC染色法测定大鼠左侧脑组织梗死面积,免疫组化检测大鼠脑组织海马区Caspase-3的表达,原位杂交检测大鼠海马组织Caspase-3 m RNA的转录水平。结果与模型组比较,丙泊酚组脑组织灰白色区域明显缩小,改善了脑组织梗死面积,海马区Caspase-3的阳性表达和Caspase-3 m RNA的转录水平明显降低(P0.01)。结论丙泊酚干预对大鼠脑缺血再灌注损伤有保护作用,其机制可能通过干预Caspase-3相关的细胞凋亡信号传导途径来完成。     

β-淀粉样肽25-35致记忆障碍大鼠中枢双孔钾通道亚型mRNA表达的改变

目的　研究聚集态 β 淀粉样肽2 5 3 5(β AP2 5 3 5)致记忆障碍大鼠模型中枢双孔钾通道亚型mRNA表达的变化。方法　Morris水迷宫实验检验大鼠空间学习记忆能力 ,用RT PCR方法检测大脑皮层和海马中 3种双孔钾通道TREK 1 ,TREK 2和TRAAKmRNA的表达。结果　在Morris水迷宫实验中 ,β AP2 5 3 5注射组大鼠d 1 ,d 2和d 4的潜伏期均比对照组显著延长。β AP2 5 3 5注射组大鼠大脑海马中TREK 1 ,TREK 2和TRAAKmRNA的表达比对照组明显升高 ;而在皮层中的表达变化不显著。结论　侧脑室注射聚集态 β AP2 5 3 5致大鼠学习记忆障碍的同时可诱发中枢双孔钾通道亚型的表达改变     

大鼠慢性脑缺血后超极化激活阳离子通道亚型HCN1 mRNA的改变

目的：进一步探讨超极化激活阳离子通道（HCN）在脑缺血损伤中的作用。方法：成年雄性SD大鼠，永久性结扎双侧颈总动脉制作慢性弥漫性全脑缺血模型。运用原位杂交及半定量RT-PCR方法，检测超极化激活阳离子通道亚型HCN1 mRNA在大脑皮层和海马的表达。结果慢性脑缺血30天后HCN1 mRNA表达明显减少。结论：超极化激活阳离子通道亚型HCN1参与脑缺血病理生理变化，其确切作用与作用机制有待深入研究.     

Down regulation of cyclooxygenase-2 is involved in delayed neuroprotection by ischemic preconditioning in rats

Aim: To examine whether the prostaglandins (PGs) pathway is involved in triggering delayed neuroprotection by ischemic preconditioning (IPC) and evaluate the effects of IPC on cyclooxygenase-2 (COX-2) expression following focal cerebral ischemia and reperfusion in rats. Methods: IPC was induced by 10min of saline infusion into the left internal carotid artery with the right common carotid artery clamped at the same time. Middle cerebral artery occlusion (MCAO) and reperfusion model was prodt:ced using intraluminal filament method. Results: IPC 48h priorto MCAO significantly reduced infarct area as compared with MCAO alone. A nonselective inhibitor of COX indomethacin (3mg/kg ip) applied 1h prior to or 1h after IPC failed to affect its protective effects. IPC had no direct effect on the cortex COX-2 mRNA and protein expression 72h later, but decreased the expression of COX-2 mRNA and protein following ischemia and reperfusion insult. Conclusion: PGs pathways was not involved in triggering delayed neuroprotection by IPC, and IPC induced down-regulation of COX-2 following focal cerebral ischemia and reperfusion in rats in vivo.     

Effects of fluoxetine on protein expression of potassium ion channels in the brain of chronic mild stress rats

The purpose of this study is to investigate the expression of major potassium channel subtypes in the brain of chronical mild stress (CMS) rats and reveal the effects of fluoxetine on the expression of these channels. Rats were exposed to a variety of unpredictable stress for three weeks and induced anhedonia, lower sucrose preference, locomotor activity and lower body weight. The protein expressions were determined by Western blot. CMS significantly increased the expression of Kv2.1 channel in frontal cortex but not in hippocampus, and the expression level was normalized after fluoxetine treatment. The expression of TREK-1 channel was also obviously increased in frontal cortex in CMS rats. Fluoxetine treatment might prevent this increase. However, the expression of Kv3.1 and Kv4.2 channels was considerably decreased in hippocampus after CMS, and was not affected by fluoxetine. These results suggest that different subtypes of potassium channels are associated with the pathophysiology of depression and that the therapeutical effects of fluoxetine may relate to Kv2.1 and TREK-1 potassium channels.KEY WORDS: Potassium ion channel, CMS, Kv2.1, TREK-1, Depression, Rat     

丁基苯酞对大脑中动脉阻断后皮层组织中花生四烯酸释放及磷脂酶A2基因表达的影响

目的　研究丁基苯酞 (dl NBP) ,d NBP和l NBP对大脑中动脉阻断 (MCAO) 6h后缺血区皮层中花生四烯酸 (AA)释放及磷脂酶A2 (PLA2 )基因表达的影响。方法　阻断大脑中动脉起始部造成局灶性脑缺血模型。HPLC检测AA。Northernblot检测皮层中PLA2 基因表达。结果　MCAO后 6h ,皮层中AA释放明显增加。于脑缺血后 5min和 12 0min ,给dl NBP(10或 2 0mg·kg- 1)和尼莫地平 (0 5mg·kg- 1)可显著抑制AA的释放。d NBP和l NBP作用比较 ,显示d NBP有与dl NBP相似的作用 ,而l NBP则无明显影响。Northern印迹结果表明 ,脑缺血 6h ,皮层中PLA2 的基因表达增强。dl NBP和d NBP(10 ,2 0mg·kg- 1,ip)皆可使表达降低 ,而l NBP对缺血脑组织中PLA2 的基因表达的升高无明显影响。结论　dl NBP和d NBP可抑制MCAO后脑组织中AA释放和PLA2 的基因表达。     

Protective effects of imperatorin against cerebral ischemia/reperfusion-induced oxidative stress through Nrf2 signaling pathway in rats

OBJECTIVE To investigates the effects of imperatorin on the oxidative stress in the cerebral cortex and hippocampus after focal cerebral ischemia/reperfusion injury.METHODS Transient focal cerebral ischemia/reperfusion model in male Sprague-Dawley rats was induced by 2 h middle cerebral artery occlusion followed by 24 h reperfusion.Imperatorin(1.25 and 2.5 mg·kg-1)or vehicle were administered intraperitoneally at 1,5 and 9 h after the onset of ischemia.At 24 h after reperfusion,the biomarkers of oxidative stress such as the levels of reactive oxygen species(ROS),lipid peroxidation products malondialdehyde(MDA),nitric oxide(NO)and total antioxidant capacity(T-AOC),the activities of inducible nitric oxide synthase(iN OS),superoxide dismutase(SOD)and catalase(CAT)in the cerebral cortex and hippocampus were observed.We also assessed the nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),and the NAD(P)H-quinone oxidoreductase 1(NQO-1)protein expression by Western blot.RESULTS As compared to vehicle-treated animals,imperatorin treatment significantly reduced the ROS,MDA,NO levels and i NOS activity,increased T-AOC and the activities of SOD and CAT.Furthermore,imperatorin treatment also significantly induced the nuclear translocation of Nrf2,enhanced the protein expression of HO-1 and NQO-1 in the cerebral cortex and hippocampus.CONCLUSION Our findings indicate that imperatorin can protect the brain against the excessive oxidative stress induced by cerebral ischemia/reperfusion through activation of Nrf2 signaling pathway.     

脑缺血再灌注损伤大鼠大脑皮质NR2A及NR2B受体表达变化

目的探讨N-甲基-D-天冬氨酸受体亚单位NR2A/2B表达与缺血再灌注损伤的关系。方法建立局灶性大脑中动脉阻塞大鼠模型观察缺血2 h再灌6～96 h的组织病理学改变,实时荧光定量PCR及Western印迹法测定大脑皮质NR2A/2B mRNA及蛋白表达。结果大鼠缺血再灌注后6 h,皮质开始出现明显病理学改变,12 h可见血管内有淤血,24 h梗死区锥体细胞出现严重的核固缩、核溶解,几乎看不到正常神经元,48 h出现大面积角质化,96 h可见炎症细胞浸润。与假手术组相比,再灌组NR2A/2B mRNA于再灌注6 h即开始一直持续明显下降(P<0.01),再灌12和24 hNR2A/2B mRNA比值均为1∶2,偏离正常的1∶1,48 h两者的表达开始上调,至96 h NR2A/2B mRNA比值达到1∶1;再灌24 h后NR2A蛋白表达显著降低(P<0.05);NR2B蛋白于再灌6 h开始明显降低,一直持续到24 h(P<0.01),48 h开始上调,96 h后蛋白表达接近假手术组水平。结论缺血2 h再灌注24 h后神经元损伤最严重,并与NR2A/2B表达改变存在时间一致性和受体亚型选择性。     

黄芪注射液对脑缺血/再灌注大鼠海马组织JNK-3表达的影响

目的观察黄芪注射液对脑缺血/再灌注大鼠海马组织JNK3蛋白及其mRNA表达的影响。方法采用4VO法制备脑缺血/再灌注大鼠模型。设假手术组、模型组、黄芪注射液组和黄芪注射液溶剂对照组。除假手术组外,其余各组缺血30 min后再灌注,根据再灌注不同时间点再分为0、0.5、2、6、24、72和120 h 7个亚组。黄芪注射液组于缺血前30 min腹腔注射黄芪注射液[12 g(生药)·kg-1],其中24、72、120 h组手术后每隔24 h追加给药1次,黄芪注射液溶剂对照组腹腔注射与黄芪注射液等量的无菌去离子水。分别采用HE染色观察组织形态学变化;免疫组织化学法和Western blot法检测大鼠海马组织JNK3蛋白表达的变化;RT-PCR方法检测海马组织JNK3 mRNA的表达。结果 HE染色结果显示黄芪注射液可改善脑缺血/再灌注造成的大鼠海马神经元损伤;除120 h外,模型组各时间点海马组织JNK3蛋白及mRNA表达均较假手术组增加(P<0.05);与模型组相比,黄芪注射液组除120 h外各时间点JNK3蛋白及mRNA表达均降低(P<0.05),而黄芪注射液溶剂对照组在各个时间点与模型组相比差异均无显著性(P>0.05)。结论黄芪注射液可抑制脑缺血/再灌注大鼠海马组织JNK3蛋白及其mRNA表达,从而抑制脑缺血/再灌注引起的大鼠海马神经元凋亡。     

氧化苦参碱对大鼠局灶性脑缺血损伤的保护作用及其抑制凋亡的作用机制

目的探讨氧化苦参碱(oxymatrine,OMT)对大鼠局灶性脑缺血损伤的保护作用及其抑制凋亡的作用机制。方法采用大鼠永久性大脑中动脉阻塞(permanent middle cer-ebral artery occlusion,pMCAO)方法,建立脑缺血模型,大鼠pMCAO术后通过腹腔给予OMT(30、60、120 mg.kg-1),以脑梗死体积、脑含水量和行为学症状等指标评价OMT对脑缺血的神经保护作用。通过HE染色方法观察OMT对缺血皮层神经细胞的形态变化以及数目的影响;应用Westernblot法检测OMT对缺血皮层Caspase-3、Bcl-2、Bax蛋白水平的表达。结果在大鼠局灶性脑缺血体内模型中,OMT(30、60、120 mg.kg-1)可明显减小脑梗死体积、脑含水量和改善行为学体征(P<0.01)。HE染色结果提示:OMT可明显增加神经细胞的存活率改善神经细胞的形态。Western blot结果显示:与假手术组相比,大鼠pMCAO后3、6、12、24 h,缺血皮层Caspase-3、Bax蛋白的表达水平在3 h开始上升,24 h达到峰值;而Bcl-2的表达水平在3h开始下降,24 h降到最低。给予OMT后可下调pMCAO大鼠缺血皮层中Caspase-3、Bax蛋白,上调Bcl-2蛋白。结论氧化苦参碱对脑缺血损伤有直接的神经保护作用,其机制可能通过上调Bcl-2及下调Bax、Caspase-3蛋白水平抑制凋亡发生。     

Novel tarantula toxins for subtypes of voltage-dependent potassium channels in the Kv2 and Kv4 subfamilies

Three novel peptides with the ability to inhibit voltage-dependent potassium channels in the shab (Kv2) and shal (Kv4) subfamilies were identified from the venom of the African tarantulas Stromatopelma calceata (ScTx1) and Heteroscodra maculata (HmTx1, HmTx2). The three toxins are 34- to 38-amino acid peptides that belong to the structural family of inhibitor cystine knot spider peptides reticulated by three disulfide bridges. Electrophysiological recordings in COS cells show that these toxins act as gating modifier of voltage-dependent K+ channels. ScTx1 is the first high-affinity inhibitor of the Kv2.2 channel subtype (IC50, 21.4 nM) to be described. ScTx1 also inhibits the Kv2.1 channels, with an IC50 of 12.7 nM, and Kv2.1/Kv9.3 heteromultimers that have been proposed to be involved in O2 sensing in pulmonary artery myocytes. In addition, it is the most effective inhibitor of Kv4.2 channels described thus far, with an IC50 of 1.2 nM. HmTx toxins share sequence similarities with both the potassium channel blocker toxins (HmTx1) and the calcium channel blocker toxin omega-GsTx SIA (HmTx2). They inhibit potassium current associated with Kv2 subtypes in the 100 to 300 nM concentration range. HmTx2 seems to be a specific inhibitor of Kv2 channels, whereas HmTx1 also inhibits Kv4 channels, including Kv4.1, with the same potency. HmTx1 is the first described peptide effector of the Kv4.1 subtype. Those novel toxins are new tools for the investigation of the physiological role of the different potassium channel subunits in cellular physiology.