NSC23766改善大鼠脑缺血后认知功能缺陷

目的:探讨Rac1在大鼠海马CA1区缺血性神经元损伤中的作用.方法:健康成年雄性SD大鼠制作四动脉闭塞全脑缺血模型,实验动物随机分为Sham、缺血再灌组(ischemia/reperfusion,I/R)、溶剂对照(Vehicle)组(I/R+生理盐水)、NSC23766组(I/R+NSC23766).采用激光共聚焦显微镜技术观察海马CA1区生存神经元.利用Morris水迷宫观察脑缺血再灌注后大鼠的空间学习记忆功能的变化情况.结果:与缺血再灌注组相比,Rac1抑制剂NSC23766组海马CA1区神经元生存数量增加;大鼠缺血后的空间学习记忆缺陷明显得到改善.结论:Rac1的激活可能是导致大鼠缺血再灌注后神经元损伤的重要因素,其抑制剂NSC23766可有效减轻这种损伤,为临床治疗缺血性脑中风提供理论依据.     

创伤后应激障碍小鼠海马恐惧记忆消退相关神经元的异常激活

目的:分析创伤后应激障碍(PTSD)小鼠恐惧记忆消退过程中海马各亚区神经元的激活及差异。方法:使用两种即刻早期基因蛋白标记策略,对PTSD小鼠条件性恐惧记忆消退相关神经元进行标记。第一组利用免疫荧光染色技术,使用野生型小鼠对海马区域神经元Arc蛋白进行标记并观察;第二组使用Fos-CreER^T2;Ai9转基因小鼠,在恐惧记忆消退前23 h注射他莫昔芬诱导荧光蛋白表达,标记相关神经元并观察。通过对标记的海马神经元数量及树突分支结构进行分析,对比海马神经元激活水平和神经元树突的可塑性变化。结果:两组实验观察发现Arc和Fos阳性神经元主要分布在背侧海马,其中Arc蛋白显色在CA3和DG亚区呈富集分布,CA1、CA2亚区的分布则较为散在,而Fos阳性神经元在海马DG亚区表达富集,散在分布于CA1、CA2和CA3亚区。与对照组对比,PTSD组海马各亚区表达Arc蛋白神经元数目没有明显差异;而PTSD组海马中CA1、CA3、DG亚区的Fos阳性神经元数量显著增加(P<0.01)。利用Fos-CreER^T2;Ai9小鼠对两组小鼠海马区域神经元树突分...     

肺炎衣原体感染通过PI3K激活Rac1而诱导血管平滑肌细胞迁移

 目的:研究Rac1活化在肺炎衣原体（C.pn）感染诱导血管平滑肌细胞（VSMCs）迁移中的作用及磷脂酰肌醇3-激酶（PI3K）对其活化的影响。方法:谷胱甘肽巯基转移酶（GST）-p21活化激酶1 p21结合结构域（PBD）即GST-PBD重组质粒转化感受态细菌,诱导融合蛋白表达并纯化;GST-pull down实验检测C.pn感染VSMCs后Rac1活性的变化;PI3K特异性抑制剂LY294002 （25 μmol/L）预处理VSMCs,GST-pull down实验检测Rac1活性的变化;Rac1特异性抑制剂NSC23766（50 μmol/L）预处理VSMCs,wound-healing 实验和Transwell 实验观察VSMCs迁移能力的变化。结果:重组质粒转化感受态细菌后,经诱导表达和纯化,得到足量有效的GST-PBD融合蛋白;GST-pull down实验结果显示,C.pn感染VSMCs后Rac1活性增强且显著高于正常对照组（P<0.05）;LY294002预处理VSMC后,C.pn感染诱导的Rac1活性明显下降（P<0.05）;细胞迁移实验结果显示,NSC23766预处理的 C.pn感染组细胞迁移能力明显低于单纯感染组（P<0.05）。结论: C.pn感染可能通过PI3K激活Rac1,从而诱导VSMCs迁移。     

不可分型流感嗜血杆菌诱导NCI-H292细胞产生MUC5AC的分子机制

目的:探讨不可分型流感嗜血杆菌(nontypeable Haemophilus influenzae,NTHi)诱导气道上皮细胞表达黏蛋白MUC5AC的影响,并探讨其可能的分子机制。方法:体外培养NCI-H292细胞,用NTHi感染后,采用ELISA检测MUC5AC和基质金属蛋白酶9(MMP-9)的水平;明胶酶谱实验分析MMP-9的酶活性;同时分别采用表皮生长因子受体(EGFR)、磷脂酰肌醇3-激酶(PI3K)、NADPH氧化酶、活性氧簇(ROS)、和MMP-9特异性抑制剂AG1478、LY294002、DPI、NAC和GM6001预处理NCI-H292细胞,检测MUC5AC以及MMP-9的水平。结果:NTHi能以时间依赖性方式诱导NCI-H292细胞产生MMP-9,并上调其酶活性,同时增加Rac1的活性并诱导ROS生成;采用AG1478和LY294002处理后,Rac1活性显著降低;采用DPI或Rac1抑制剂NSC23766处理后,ROS含量明显减少;当NCI-H292细胞用NAC或NSC23766预处理后,可显著下调MMP-9的表达与活性。此外,采用GM6001处理后,MUC5AC的分泌明显降低。结论:NTHi经EGFR/PI3K/Rac1/NADPH氧化酶/ROS/MMP-9通路诱导NCIH292细胞产生MUC5AC。     

促红细胞生成素上调海马pCREB表达和改善脑缺血小鼠认知功能

目的: 探讨促红细胞生成素(EPO)对小鼠脑缺血所致的认知功能障碍和海马神经元损伤的保护作用及机制。方法: C57BL/6绿色荧光蛋白转基因小鼠随机分为假手术(sham)组、脑缺血/再灌注(I/R)组和EPO治疗组;采用双侧颈总动脉阻断(2-VO)方法复制小鼠全脑缺血模型,跳台实验测试小鼠学习记忆能力,Nissl染色检测海马神经元存活情况,Western blotting检测磷酸化cAMP反应元件结合蛋白(pCREB)表达水平,激光共聚焦显微镜和Neurolucida软件分析检测海马CA1区神经元形态及树突棘的变化。结果: 脑缺血导致小鼠学习记忆能力下降,海马CA1区神经元迟发性死亡和树突棘丢失;EPO治疗能显著提高脑缺血小鼠的学习记忆能力,减少脑缺血所致的海马CA1区神经元死亡和树突棘的丢失,显著上调海马CA1区神经元pCREB蛋白的表达。结论: EPO可能通过上调pCREB的表达来保护神经元损伤、防止神经元树突棘的丢失,进而改善脑缺血小鼠的认知功能。     

APP/PS1转基因AD鼠海马CA1区神经元突触超微结构

目的:探讨携带5个家族性基因突变的APP/PS1转基因阿尔茨海默病(AD)(5×FAD)模型小鼠海马CA1区神经元突触超微结构改变。方法:应用透射电镜观察和形态计量学分析5×FAD转基因AD鼠海马CA1区GrayⅠ型突触界面结构参数,包括突触间隙长度、突触间隙面积、突触后致密物浓度和突触界面曲率的变化。结果:5×FAD转基因AD鼠海马CA1区神经元突触活性区长度显著小于对照组,差异有统计学意义;突触后致密物厚度、突触界面曲率及宽度与对照组差异无统计学意义。结论:5个家族性突变基因导致小鼠海马CA1区神经元突触可塑性的改变,这可能是该突变基因导致的发病机制之一。     

神经病理性痛大鼠海马CA1区锥体神经元树突及树突棘的变化

目的:观察神经病理性痛条件下大鼠海马CA1区锥体神经元树突形态和树突棘密度的变化。方法:建立大鼠腰5脊神经结扎(L5 spinal nerve ligation,SNL)神经病理性痛模型,对照组为假手术组即只暴露腰5脊神经,不结扎。利用Von Frey纤维丝检测其50%机械性缩足阈值(paw withdrawal threshold,PWT),判断SNL模型是否成功。通过高尔基(Golgi)染色的方法观察SNL模型后14 d海马CA1区锥体神经元树突形态和树突棘密度的变化。结果:SNL模型组大鼠CA1区锥体神经元树突棘密度升高,与对照组相比有统计学差异(P0.05),而锥体神经元树突分支数无明显差异。结论:神经病理性痛会导致海马CA1区锥体神经元树突棘密度升高。     

抑制Rac1通过降低磷酸化p38MAPK提高1型糖尿病小鼠的心脏功能

目的:探讨抑制Rac1对1型糖尿病小鼠心肌细胞肥大、心脏功能的影响及其作用机制。方法:50只8周龄雄性C57小鼠随机分为对照组(control,n=10)、Rac1抑制剂NSC23766对照组(NSC,n=10)、1型糖尿病组(STZ,n=15)及NSC治疗组(STZ+NSC,n=15)。小鼠腹腔注射链脲佐菌素(STZ)建立1型糖尿病动物模型,血糖升高后给予小鼠腹腔注射NSC23766,实验于8周末结束,记录实验小鼠生存率、测量小鼠体重及左室重量并计算左室重量指数,运用超声心动图检测小鼠心脏功能,心肌组织进行HE染色结合图像分析软件定量心肌细胞大小,运用实时定量RT-PCR技术检测心肌组织心房利钠肽(ANP)、脑利尿肽(BNP)、β-肌球蛋白重链(β-MHC)mRNA表达,以Westernblotting定量心肌组织磷酸化p38丝裂素活化蛋白激酶(pho-p38MAPK)表达。结果:抑制Rac1后:(1)糖尿病小鼠生存率提高、糖尿病小鼠左室重量指数降低(P0.01)、心脏左室射血分数(EF)增加、左室短轴缩短率增加(FS)(P0.01);(2)心肌细胞大小明显降低(P0.01);(3)心肌肥大相关基因ANP、BNP、β-MHC表达明显降低(P0.01);(4)心肌组织pho-p38MAPK表达明显降低(P0.01)。结论:抑制Rac1活性显著改善1型糖尿病小鼠心脏功能、降低心肌细胞肥大,其机制可能与明显降低心肌组织磷酸化p38MAPK密切相关。     

小鼠海马CA1区锥体神经元树突棘的发育

目的 对小鼠海马CA1区锥体神经元正常发育中树突棘密度及各种形态变化进行分析测定,为深入研究突触发生及突触可塑性提供直接的形态学依据.方法 分别取出生后0、5、10、20及30d 5个年龄段的C57BL/6小鼠各10只,采用基因枪对小鼠海马CA1区锥体神经元树突棘进行亲脂性荧光染料DiI标记,通过激光共焦显微镜对其进行观察分析;同时利用透射电镜技术对树突棘的超微结构进行分析.结果 树突棘的形态、大小及其密度随小鼠发育而变化,成熟树突棘内部存在滑面内质网与棘器,可能参与了突触后膜结合蛋白及其转运体的合成.结论 树突棘的发育过程与突触连接的形成以及突触可塑性密切相关.     

急性饮酒对小鼠海马 CA1区锋电位的影响

观察研究急性饮酒前后,小鼠海马区锋电位（Spike）的放电频率及峰峰间期等的变化,描述并分析清醒状态及急性饮酒后小鼠海马区神经元电信号的特征差异,评价急性饮酒对小鼠海马区记忆功能的影响。以 ICR 小鼠为实验对象,分成急性饮酒组（P 组）和生理盐水对照组（C 组）。在小鼠海马 CA1区植入8通道微电极阵列,利用神经信号处理采集系统 Cerebus 记录急性饮酒前后小鼠海马 CA1区神经电信号,分析比较海马区神经元放电频率以及锋电位间隔直方图（Interspike Interval Histograms ,ISI）的变化。P 组与 C 组比较,P 组平均放电率小于 C 组;与清醒状态下比较,急性饮酒后小鼠海马区神经元放电频率变低,然后随着时间逐渐增强,慢慢恢复;ISI 值从较为集中变为较为分散。急性饮酒后小鼠海马区神经元自发放电在放电频率、ISI 等神经信号的特征方面存在明显差异。急性饮酒可抑制小鼠海马区神经元放电,抑制小鼠记忆功能。     

Activation of phosphatidylinositol 3-kinase/Akt-mammalian target of Rapamycin signaling pathway in the hippocampus is essential for the acquisition of morphine-induced place preference in rats

Hippocampus is a critical structure for the acquisition of morphine-induced conditioned place preference (CPP), which is a usual learning paradigm for assessing drug reward. However, the precise mechanisms remain largely unknown. Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt, mammalian target of Rapamycin (mTOR) and 70-kDa ribosomal S6 kinase (p70S6K), are critical molecules implicated in learning and memory. Here, we tested the role of PI3K/Akt-mTOR-p70S6K signaling pathway in morphine-induced CPP in the hippocampus. Our results showed that the acquisition of morphine CPP increased phosphorylation of Akt in the hippocampal CA3, but not in the nucleus accumbens (NAc), the ventral tegmental area (VTA) or the CA1. Moreover, the phosphorylated Akt exclusively expressed in the CA3 neurons. Likewise, levels of phosphorylated mTOR and p70S6K were significantly enhanced in the CA3 following morphine CPP. The alterations of these phosphorylated proteins are positively correlated with the acquisition of morphine CPP. More importantly, microinjection of PI3K inhibitor (LY294002) or mTOR inhibitor (Rapamycin) into the CA3 prevented the acquisition of CPP and inhibited the activation of PI3K-Akt signaling pathway. In addition, pre-infusion of β-FNA (β-funaltrexamine hydrochloride), a selective irreversible μ opioid receptor antagonist, into CA3 significantly prevented the acquisition of CPP and impaired Akt phosphorylation. All these results strongly implied that the PI3K-Akt signaling pathway activated by μ opioid receptor in hippocampal CA3 plays an important role in acquisition of morphine-induced CPP.     

Rac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in human blood stimulated by atherosclerotic plaque

Background  

Platelet activation requires rapid remodeling of the actin cytoskeleton which is regulated by small GTP-binding proteins. By using the Rac1-specific inhibitor NSC23766, we have recently found that Rac1 is a central component of a signaling pathway that regulates dephosphorylation and activation of the actin-dynamising protein cofilin, dense and α-granule secretion, and subsequent aggregation of thrombin-stimulated washed platelets.     

Retrieval of contextual memories increases activity-regulated cytoskeleton-associated protein in the amygdala and hippocampus

Activity-regulated cytoskeleton-associated protein (Arc) integrates information from multiple intracellular signaling cascades and, in turn, regulates cytoskeletal proteins involved in structural synaptic modifications. The purposes of the present study were: (1) to determine if the retrieval of contextual memories would induce Arc in hippocampal and amygdalar neurons; (2) use unbiased stereology at the ultrastructural level to quantify synapses contacting Arc-labeled (Arc+) and unlabeled (Arc?) postsynaptic structures in brain regions in which the amount of Arc integrated density (ID) correlated strongly with the degree of amphetamine conditioned place preference (AMPH CPP). The retrieval of contextual memories increased the Arc ID in the dentate gyrus, cornu ammonis (CA)1, and CA3 fields of the hippocampus and the basolateral, lateral, and central nuclei of the amygdala but not the primary auditory cortex, a control region. Stereological quantification of Arc+ and Arc? synapses in the basolateral nucleus of the amygdala (BLA) was undertaken because the strongest relationship between the amount of Arc ID and AMPH CPP was observed in the BLA. The retrieval of contextual memories increased the number and density of asymmetric (presumed excitatory) synapses contacting Arc+ spines and dendrites of BLA neurons, symmetric (presumed inhibitory or modulatory) synapses contacting Arc+ dendrites of BLA neurons, and multisynaptic boutons contacting Arc+ postsynaptic structures. Thus, the retrieval of contextual memories increases Arc in the amygdala and hippocampus, an effect that could be important for approach behavior to a drug-associated context.     

Foreign body giant cell formation is preceded by lamellipodia formation and can be attenuated by inhibition of Rac1 activation

Macrophages that are recruited to the site of implanted biomaterials undergo fusion to form surface-damaging foreign body giant cells. Exposure of peripheral blood monocytes to interleukin-4 can recapitulate the fusion process in vitro. In this study, we used interleukin-4 to induce multinucleation of murine bone marrow-derived macrophages and observed changes in cell shape, including elongation and lamellipodia formation, before fusion. Because cytoskeletal rearrangements are regulated by small GTPases, we examined the effects of inhibitors of Rho kinase (Y-32885) and Rac activation (NSC23766) on fusion. Y-32885 did not prevent cytoskeletal changes or fusion but limited the extent of multinucleation. NSC23766, on the other hand, inhibited lamellipodia formation and fusion in a dose-dependent manner. In addition, we found that in control cells, these changes were preceded by Rac1 activation. However, NSC23766 did not block the uptake of polystyrene microspheres. Likewise, short interfering RNA knockdown of Rac1 limited fusion without limiting phagocytosis. Thus, phagocytosis and fusion can be partially decoupled based on their susceptibility to NSC23766. Furthermore, poly(ethylene-co-vinyl acetate) scaffolds containing NSC23766 attenuated foreign body giant cell formation in vivo. These observations suggest that targeting Rac1 activation could protect biomaterials without compromising the ability of macrophages to perform beneficial phagocytic functions at implantation sites.     

Inhibition of Rac1 decreases the severity of pancreatitis and pancreatitis-associated lung injury in mice

Pancreatitis is a disease with high morbidity and mortality. In vitro experiments on pancreatic acini showed that supramaximal but not submaximal cholecystokinin (CCK) stimulation induces effects in the acinar cell that can be correlated with acinar morphological changes observed in the in vivo experimental model of cerulein-induced pancreatitis. The GTPase Rac1 was previously reported to be involved in CCK-evoked amylase release from pancreatic acinar cells. Here, we demonstrate that pretreatment with the Rac1 inhibitor NSC23766 (100 microM, 2 h) effectively blocked Rac1 translocation and activation in CCK-stimulated pancreatic acini, without affecting activation of its closely related GTPase, RhoA. This specific Rac1 inhibition decreased supramaximal (10 nM) CCK-stimulated acinar amylase release (27.% reduction), which seems to be connected to the reduction observed in serum amylase (46.6% reduction) and lipase levels (46.1% reduction) from cerulein-treated mice receiving NSC23766 (100 nmol h(-1)). The lack of Rac1 activation also reduced formation of reactive oxygen species (ROS; 20.8% reduction) and lactate dehydrogenase release (LDH; 24.3% reduction), but did not alter calcium signaling or trypsinogen activation in 10 nM CCK-stimulated acini. In the in vivo model, the cerulein-treated mice receiving NSC23766 also presented a decrease in both pancreatic and lung histopathological scores (reduction in oedema, 32.4 and 66.4%; haemorrhage, 48.3 and 60.2%; and leukocyte infiltrate, 53.5 and 43.6%, respectively; reduction in pancreatic necrosis, 65.6%) and inflammatory parameters [reduction in myeloperoxidase, 52.2 and 38.9%; nuclear factor kappaB (p65), 61.3 and 48.6%; and nuclear factor kappaB (p50), 46.9 and 44.9%, respectively], together with lower serum levels for inflammatory (TNF-alpha, 40.4% reduction) and cellular damage metabolites (LDH, 52.7% reduction). Collectively, these results suggest that pharmacological Rac1 inhibition ameliorates the severity of pancreatitis and pancreatitis-associated lung injury through the reduction of pancreatic acinar damage induced by pathological digestive enzyme secretion and overproduction of ROS.     

Prefrontal cortex lesions differentially disrupt cocaine-reinforced conditioned place preference but not conditioned taste aversion

The reinforcing efficacy of cocaine is thought to involve, at least in part, mesocortical dopaminergic (DA) neurons. Rats will self-administer cocaine applied directly into the medial prefrontal cortex but not into nucleus accumbens or the ventral tegmental area (Goeders & Smith, 1983). The present experiments were conducted to assess whether lesions of prefrontal cortex (mesocortical DA target regions) attenuate the reinforcing properties of systemically administered cocaine. Male Sprague-Dawley rats were anesthetized, and one of three subfields (medial, orbital, or precentral) of the prefrontal cortex was removed by aspiration or no brain injury was done (sham operates). In four experiments the rats were tested on conditioned place preference (CPP), conditioned taste aversion (saccharin conditioned stimulus, cocaine unconditioned stimulus), general activity in the running wheel and open field, and food-reinforced spatial alternation in the T-maze. Sham operates demonstrated a cocaine-induced place preference, rats with medial frontal lesions showed a cocaine-induced place aversion, and other operates showed neither a conditioned place preference nor an aversion. The results of this experiment suggest that lesions of the DA projection fields of the prefrontal cortex in the rat reduce the positive reinforcing properties of systemically injected cocaine. In the second experiment, all subjects showed a conditioned taste aversion of equal magnitude. This suggests that whereas the positive reinforcing properties were affected differentially by prefrontal cortex lesions, the aversive properties were not affected. In Experiment 3 there were no lesion-induced differences in activity in either the running wheel or the open field. Therefore, changes in motor activity cannot account for the CPP data. In the final experiment, the medial frontal operates were impaired relative to the precentral and sham operates on learning to alternate choices in the T-maze, but the orbital frontal operates' performance was not different from that of any other group. This suggests that a general disruption of all reinforcement mechanisms did not occur following these lesions. Instead, these results indicate that mesocortical DA projection regions are involved with mediating the reinforcing properties of cocaine and that there is a separate system mediating the aversive properties of cocaine.     

Pre-treatment with high doses of cocaine decreases the reinforcing effects of cocaine in the conditioned place preference paradigm

The aim of the present study was to determine if pre-exposure to high doses of cocaine can subsequently alter the rewarding effects of this drug. Adult male mice received a pretreatment of physiological saline, or 12.5 or 25 mg/kg of cocaine (one injection a day for five days). After an interval of six days without injections, the rewarding effects of low doses of cocaine (0.5, 1 or 1.5 mg/kg) were evaluated in the conditioned place preference (CPP) paradigm. Doses of 1 and 1.5 mg/kg induced a clear CPP in animals pre-treated with saline but were ineffective in those pre-treated with 25 mg/kg of cocaine. Only the dose of 1.5 mg/kg induced CPP in mice pre-treated with 12.5 mg/kg of cocaine. Our results, which reveal a decrease in the conditioned rewarding effects of threshold doses of cocaine, demonstrate that exposure to high doses of this drug can alter the reward system.     

18-Methoxycoronaridine blocks acquisition but enhances reinstatement of a cocaine place preference

The iboga alkaloid congener, 18-methoxycoronaridine (18-MC), decreases self-administration of multiple drugs of abuse. Here, in a biased procedure, we investigated whether 18-MC would have a similar effect on the acquisition, expression and reinstatement of a cocaine conditioned place preference (CPP) in male Sprague-Dawley rats. While 18-MC attenuated acquisition of a cocaine CPP, it had no effect on CPP expression, and enhanced the reinstatement of cocaine CPP following extinction. Our results are consistent with those obtained using ibogaine, but reinforce the notion that acquisition, expression and reinstatement of a CPP likely involve separate mechanisms.