氯胺酮与乙醇对小鼠海马PKA-CREB 信号通路的影响

南通大学学报(医学版Journal of Nantong University(Medical Sciences) 2013: 33(4283氯胺酮与乙醇对小鼠海马PKA-CREB信号通路的影响杨美玉13”,丁飞2,蒋小岗3,顾振纶3,卞士中”(苏州大学医学部法医学系,苏州215123;2上海市公安局宝山分局刑事科学研究所;3苏州中药研究所)摘要]目的:研究氯胺酮与乙醇对小鼠海马ⅣKA-CREB信号通路的影响,探讨氯胺酮与乙醇致学习记忆障碍的分子学机制。方法:40只ICR小鼠随机分为4组:对照组、氯胺酮组、乙醇组、氯胺酮乙醇合用组,每组10只。氯胺酮和乙醇分别釆取腹腔注射和灌胃的给药方式,1次/d,连续14d。通过RT-PCR检测海马中c-AMP反应单元结合蛋白(c- AMP response element binding protein,CREB)蛋白激酶A( protein kinase A,FKA)及腺苷酸环化酶( adenylate cyclase,AC)mRNA的表达; Western blot检测 PCREB/CREB的蛋白表达。结果:RT-PCR检测结果:氯胺酮乙醇合用组可显著降低海马区CREB、PKA及 AC mRNA的表达(P<0.05), Western Blot检测显示 PCREB/CREB的蛋白表达显著降低(P<0.05)。結论:氯胺酮与乙醇可协同抑制小鼠的学习记忆行为,其机制可能与大脑海马区CREB信号通路相关[关键词]氯胺酮;乙醇;学习记忆;环磷酸腺苷反应结合蛋白;小鼠中图分类号]R971文献标志码]A[文章编号]1674-7887(2013)04-0283-05Effects of ketamine on ethanol-induced learning and memory impairment in miceYANG Meiyu., DING Fei JIA NG Xiaogang, GU Zhenlun, BlA N Shizhong (Department of Forensic MedicineMedical College of Soochow University, Institute of Forensic Sciences of Soochow University, Suzhou 215123; Institute ofCriminal Sciences, Baoshan Branch of Shanghai Public Security Bureau; Suzhou Institute of Chinese Materia Medica)[Abstract] Objective: To study the effects of ketamine and ethanol on PKA-CREB pathway of hippocampus -dependentlearning and memory in mice and its possible mechanism. Methods: A total of 40 male mice were divided into 4 groups: anormal control group, a ketamine group, an ethanol group and an ethanol plus ketamine group. Ketamine and ethanol wererespectively taken by intraperitoneal injection and oral administration, once per day for 14 d. The expressions of CreBPKA and AC gene in hippocampus were detected by RT-PCR. The expressions of pCREB/CREB were detected by WesternBlot. Results: After the combined treatment with ketamin and ethanol. CREB. PKA and AC mRNA were down regulated byRT-PCR(P<0. 05), and p CREB/CREB were down regulated by Westem Blot(P<0. 05). Conclusion: The data indicatethat combination of ketamine and ethanol is synergic in the inhibition of learning and memory in mice, its mechanism maybe related to the CreB signal transduction pathway.[Key words] ketamine; ethanol; learning and memory; C-AMP-response element binding protein; mouse氯胺酮是N-甲基D-天(门)冬氨酸(N- methy-人细胞核磷酸化CREB,然后激活TEGs(C-os)等,其D- -aspartate,NMDA)受体的非特异性阻断剂,其作激活产物进一步编码LTP所需要的蛋白质。用机制复杂。目前研究认为,麻醉剂量的氯胺酮本研究以小鼠为实验对象,观察氯胺酮与酒精可通过阻断NMDA受体抑制学习记忆,该受体对神联合给药对小鼠学习记忆的影响。采用RT-PCR技经元突触长时程增强(ong- -term potentiation,TP)的术检测小鼠海马中CREB、PKA及腺甘酸环化酶产生、维持和学习记忆功能至关重要。adenylate cyclase, AC)mRNA的表达; Western blot环磷腺苷 (cyclin adenosine monophosphate,c-AMP)检测 pCREB/CREB的蛋白表达情况,以探讨氯胺酮反应元件结合蛋白(c- AMP response element binding与乙醇对小鼠学习记忆行为学影响的分子机制。protein,CREB)是一种依赖c-AMP的转录因子,可将细胞内的c-AMP的量的变化转录为基因表达的1材料与方法形式。CREB在记忆的形成过程中发挥了及其重要1.1实验动物ICR小鼠40只,雄性,体质量25的作用,其过程可能是:神经递质等作用神经元细30g。苏州大学实验动物中心提供。实验前小鼠适应胞,导致胞质内c-AMP浓度升高,使蛋白激酶A性饲养3d,维持室温(22±1)℃,在整个实验过程中protein kinase A,PKA)激活,PKA的催化亚单位进小鼠自由饮水和进食。*作者简介杨美玉,女,汉族,生于1984年3月,山东省济宁市人,硕土在读,研究方向:法医毒理病理学通信作者卞士中,电话:0512-67580893,E- mail. bianshizhongt@ suda. edu. en南通大学学报(医学版)2013:33(4)1.2主要试剂及仪器盐酸氯胺酮注射液(国药准表2内参和目的基因扩增条件字H32022820,批号:KH071004,2ml:0.1g)购自江基因扩增条件苏恒瑞医药公司;56%v/红星二锅头酒购自北京红 GAPDH94℃4min94℃30s-55℃30s72℃星股份有限公司;PCR扩增试剂盒(批号:DR0A)63lmin+72℃10min+4℃购自 Takara公司;RNA逆转录引物:由上海生物工℃5min→94℃1min→+57℃30s-72℃程有限公司合成;兔抗鼠 phospho-CREB-1(Ser13345s72℃10min*4℃抗体(一抗)(1:100)购自 slabs;抗小鼠CREB抗体94℃4min→→94℃30s57℃30s+72℃30 cycles)lmin+72℃10min-+4℃(一抗)(1:100购自武汉博士德生物技术有限公司CREB94℃2min+94℃30s→55℃45s72℃actin(1:1000)购自 Santa cruze;所用抗鼠和抗兔荧(30 cyeles)45s+72℃7min-+4℃光二抗均为 Odyssey公司产品1.5 Western blot1.3模型制备与给药40只小鼠随机分成4组,分1.5.1蛋白样品制备取液氮内冻存的小鼠海马组别为正常对照组,氯胺酮组,乙醇组,联合给药组,每织约30mg,加入组织裂解液05m,超声匀浆后于组10只。给药方式:对照组以生理盐水腹腔注射+生4℃,12000 r/min离心10min,吸取上清,分装,另理盐水灌胃,乙醇组以生理盐水腹腔注射+乙醇灌外留少许待测蛋白溶液(于-40℃冷藏。胃,氯胺酮组以氯胺酮腹腔注射+生理盐水灌胃,联1.5.2BCA试剂盒检测蛋白浓度(1)蛋白标准曲合给药组以氯胺酮腹腔注射+乙醇灌胃。生理盐水、线绘制取标准蛋白液,用PBS梯度稀释成4、8、12、氯胺酮溶液(50mgkg)腹腔注射1次l,连续14d;生16、20、24μg/μL蛋白溶液,每组40μL。取BCA试理盐水、乙醇红星二锅头稀释至20%灌胃1次,剂盒A液和B液按50:1配成工作液。稀释后的标连续14d。各组小鼠每日9:00给药。准蛋白液与BCA工作液按1:8混合均匀后,37℃水1.4PCR检测浴30min,于酶标仪上测定OD值,每组设3复孔。1.4.1总RNA提取取液氮内冻存的小鼠海马组由OD值,绘制蛋白标准曲线;(2)待测蛋白浓度的测织约30mg,加人Tiol试剂0.5mL,按说明书进行定取待测蛋白液每组10μL。将待测蛋白液与BCARNA提取,所用RNA的D/D2均在1.8~2.0。工作液按1:8混匀后,37℃水浴30min,于酶标仪上1.4.2模板cDNA合成提取的总RNA逆转录合测其OD值,每组设3复孔。根据OD值,于蛋白标成cDNA。准曲线上计算相关蛋白浓度。根据蛋白浓度测定值1.4.3 RT-PCR调整样本上样量,使各组蛋白量为60μg,按4:1与14.3.1内参和目的基因的引物序列及产物长度5× padding Buffer混匀,水浴100℃变性5min后,见表1。冰浴备用4.3.2内参和目的基因扩增条件均为25μL1.5.3SDS-PAGE电泳先将实验用玻片洗净,再反应体系,应用 GAPDH作为参照。内参和目的基用无水乙醇脱脂,晾干后组装制胶板。配12%的分离因扩增条件见表2。PCR产物用1.5%琼脂糖凝胶胶,注入制胶板中,加适量无水乙醇封闭。待胶凝固,进行电泳,结束后于全自动凝胶成像分析系统进倒去乙醇,用蒸馏水清洗3次。配制浓缩胶,两端交行灰度扫描。以 GAPDH扩増产物校正作光密度相替加入制胶板,插入梳子后放置约1h待胶凝固后对分析。使用。安装电泳装置,上样品,进行电泳,浓缩胶时设表1内参和目的基因的引物序列及产物长度置电压90V,染料条带至分离胶时电压设置至基因引物110V,染料接近分离胶底部,停止电泳。F5'-CCGGAACATGGACCTCTACTAC-31.54转膜将丙烯酰胺凝胶从玻璃板上剥离,置(284 bp) R5'-ATAGGTGGGAGGAGATGGACTT-3入转移缓冲液中,将海绵、滤纸、凝胶、NC膜、滤纸F5 '-GGAGAGCGTGAAAGAGTTCCTA-3海绵按顺序叠加,用玻璃棒小心赶走气泡,放置于转(339 bp) R5'-CCAGCTACATACTCCATGACCA-3移电泳槽中恒流400mA,转移120mi。转膜后,CREBF5=AGTATGCACAGACCACTGATGG-3用1×TBST洗膜。(392 bp) R5'-TCCACCGTAACAGGAGAGAACT-31.5.5封闭1×TBST洗膜3次,每次10min,然后GAPDH F5 -GCCATCAACGACCCCTTCATT-3将膜浸于5%脱脂牛奶中封闭1h(413 bp) R5'-CGCCTGCTTCACCACCTTCTT-31.5.6抗体孵育封闭结束后再用1×TBST洗膜3杨美玉,等氯胺酮与乙醇对小鼠海马PKA-CREB信号通路的影响285次,每次l0mins将NC膜放入小塑料袋内,加入5%的降低用统计学意义(P<0.05,图2)。提示:氯胺酮脱脂牛奶按比例稀释后的_抗,膜。去除袋内饮酒、氯胺酮+饮酒联合给药均可下调 pCREB气泡,用封膜机封口,置于摇床上室温孵育3h过CREB蛋白表达,且联合用药组 pCREB/CREB降低夜。取膜,用1×TBST洗涤3次,每次10min后,用更明显5%脱脂牛奶按1:10000的比例稀释的二抗封闭,置对照组乙醇氯胺酮氯胺酮+乙醇于摇床上,室温避光孵育1h。取膜,用1×TBST在暗pCREB-盒中洗涤3次,每次10min。15.7显影近红外双色激光成像系统显影CREB-42k1.6图像处理及数据分析RT-PCR, Western blot图像扫描后均采用 SigmaScan pro5软件系统分析,图2氯胺酮与乙醇对小鼠海马CREB, PCREB蛋白表达的实验数据以x±s表示。显著性检验采用 One way影响ANOVA,P<0.05为差异有统计学意义。3讨论2结果CREB是碱性氨基酸亮氨酸(Leu)拉链转录因子2.lRT-PCR检测结果在连续给药14d,小鼠海家族,受细胞内多条信号通路的调节,与生长抑素马组织RT-PCR检测结果分别以PKA、AC、CREB基因的cAMP反应元件 CAMP response element,CRE与内参基因 GAPDH的密度比值表示组织中PKA、有高度亲和力。CREB家族在结构上具有DNA结AC、 CREB MRNA的相对表达强度。实验结果表明,合结构域和转录激活结构域,因而具有DNA结合活与正常对照组相比,饮酒、氯胺酮、氯胺酮+饮酒联合性和转录调节活性。但只有当Ser133被PKA、蛋白给药可诱导小鼠海马组织中的RKA、AC、CREB激酶 C(protein kinase C,ⅨKC或钙/钙调蛋白激酶Ca27mRNA表达下调均P0.05。与单用药组相比,联合 calmodulin- dependent protein kinase, Ca+/CaMK等磷给药组小鼠海马组织pKA、AC、 CREB mRNA表达酸化后才能启动转录,磷酸化的CREB形成二聚体显著下降(均P<0.05图1)。提示:饮酒、氯胺酮、氯后活化,并与靶基因CRE序列结合,调节其转录,发胺酮+饮酒联合给药可使小鼠海马组织中PKA、AC、挥多种生物学效应。CREB及其相应的基因表达下调,其中氯胺酮+饮酒在中枢神经系统中,CREB调节神经细胞的生联合给药组作用更明显。长发育及营养,参与神经细胞突触可塑性和长时程Marker对照组乙醇氯胺酮氯胺酮+乙醇记忆的形成,是长时程记忆和LTP所必需的基因开关閃。CREB通过调控目的基因的表达,影响晚时(413bp相的LTP和长时程记忆实验证实CREB是学习记忆中一个关键因子参与海马的空间记忆调节。研究认为迷宫实验(284bp练可使大鼠海马和纹状体内 PCREB免疫活性显著500bp升高。海马CREB功能降低,可导致空间记忆功能的400bp减退。谭蕾等研究结果表明氯胺酮通过下调大鼠海马神经元 pCREB水平,诱导新生大鼠海马神经元凋亡,突触形成受损。 S C Pandey等研究发现大鼠(392bp)图1氯胺酮与乙醇对小鼠海马PKA、AC、CREB基因表达的过度饮酒,可导致杏仁核CRE-DNA结合力下降,海影响马内总CREB及 pCREB表达下调,进而影响大鼠的2.2 Western blot检测结果CREB的磷酸化水平学习记忆功能。与学习记忆密切有关。连续给予氯胺酮、饮酒、联合氯胺酮为苯环己哌啶(N-1- phenyeyc lohexy-给药14d后,与正常对照组相比,各用药组小鼠海 piperidine,PCP)的衍生物,是NMDA受体的非特异马组织 PCREB、CREB蛋白表达量均降低(P<0.05),性阻断剂。氯胺酮拮抗NMDA受体,可通过阻断各且磷酸化水平 pCREB/CREB的降低有统计学意义刺激产生的长时程增强效应,干扰学习记忆过程。有P005与单用药组相比联合用药组 PCREB/CREB报道称氯胺酮可致其滥用者记忆的长久损害作用。南通大学学报(医学版)2013:33(4)经多次氯胺酮麻醉的大鼠其空间学习记忆能力明显小鼠学习记忆的影响[中国药理学通报,199,15(3)下降,且海马神经元超微结构也有异常改变,损害程90-93度与使用氯胺酮的剂量水平有关叫。本实验室研究结5 Molkentin JI, Dorn gW. Cytoplasmic signaling pathways在海马内,乙醇通过抑制NMDA受体亚型2001,636330小 hypertrophy. Annu rey physiol果表明,氯胺酮多次给药可致小鼠空间学习记忆能that regulate cardiac力障碍。Wu GY, Deisseroth K, Tsien RW. Activity -dependentNR2B的活性来抑制NMDA受体。早期研究表明CREB Phospheof a fast. sensitivecalmodulin kinase pathway and a slow, less sensitive mite乙醇在较低浓度时就可对NMDA受体的一个亚型gen-activated protein kinase pathway J. Proe Natl Acad起作用,以某种方式改变该受体亚型之间的相互配Seius a.2001,985)2808-2813合,从而使谷氨酸利用受体进行的信号传递受到破7] Setlow b, Roozendaal b. McGaugh JI. Involvement of a坏,进而影响包括LTP在内的分子信号传导通路受basolateral amygdala complex-nucleus accumbens pathway阻,导致学习记忆功能障碍。体外研究证明,乙醇in glucocorticoid-induced modulation of memory consoli可明显降低CREB的活性。dation. Eur J Neurosci, 2000, 12(1): 367-375本研究分子生物学实验硏究结果表明:氯胺酮、18] Puzo d.iolo. Trinchese h,etal. Amyloid- -beta pep乙醇、氯胺酮联合乙醇作用小鼠14d后,小鼠海马ide inhibits activation of the nitric oxidelegMPicAMP-re组织AC、PKA、 CREB MRNA的表达减少,且两两比onsive element-binding protein pathwaypocampal synaptic plasticity [J). J Neurosci较显示联合用药组与单用药差异均有统计学意义6887-6897提示氯胺酮和乙醇可能在基因转录水平上对CREBLiu ry fioravante d. shah S. et al. cAMP信号传导通路协同抑制。 Western blot检测结果表ment-binding protein 1 feedback loop is necessary for明:用药14d后,小鼠海马组织中, pCREB/CREB蛋onsolidation of long-term synaptic facilitation in aplysiaJI白表达也有所下降,提示氯胺酮和乙醇在蛋白质水J Neurosci.,2008,28(8):1970-1976平上影响CREB信号传导通路。与本实验室行为学10 Colombo PJ, Brightwell JJ. Countryman RA. Cognitive str结果一致,提示氯胺酮、乙醇可加强抑制小鼠海马组ategy -specific increases in phosphorylated cAMP re-织CREB信号传导通路。sponse element-binding protein and c-Fos in the hip.CREB参与成瘾和学习记忆的形成,但还不能and dorsal striatum[J]. J Neurosci, 2003, 23(8)3547-3554仅用CREB解释行为改变的长久性。药物抑制和经m1谭蕾,罗爱林,王金韬,等氯胺酮对大鼠海马神经元验诱导的CREB活性相对短暂,且注射pCRE只能CREB磷酸化水平的影响J中华麻醉学杂志,2008部分促进突触的刺激。这表明成瘾和记忆的长时转7637-639变可能启动更加稳定调节机制,有其他因子的参与。12] Pandey so., Mittal n. Lumeng L, et al. 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J Neural Transm, 2005, 112(8)南通大学学报(医学版Journal of Nantong University(Medical Sciences) 2013: 33(4Wnt5a在严重烫伤大鼠早期肠黏膜组织中的表达及胰岛素的调控作用*俊”,胡克苏,张云,张逸(南通大学附属医院烧伤整形科,南通226001)摘要]目的:研究在严重烫伤大鼠肠黏膜组织中Wn5a的动态表达变化及其意义。方法:将48只大鼠背部浸人恒温水浴盆(92℃中20s达Ⅲ度烫伤,制作烫伤创面,采用随机数字法将大鼠分为对照组、单纯烫伤组烫伤组)、烫伤后胰岛素治疗组(治疗组)。伤后取肠组织观察大体变化,取肠道黏膜H染色后,行组织形态学观察,分别于烫伤后6、1224、48h观察,取肠黏膜组织以免疫组织化学的方法,检测Wnt5a的表达情况。结果:在烫伤组和治疗组Wnt5a表达均有显著表达,对照组有少量表达(P<0.05),伤后6-12hWn5a的表达持续升高,烫伤组12-24h开始表达逐渐减少,24h后表达又重新增多,各组在48h表达达到最高峰;与烫伤组相比,治疗组wnt5a的表达水平明显降低(P<0.05,病理形态学变化显示烫伤早期大鼠肠黏膜组织损伤明显,治疗组大鼠肠黏膜组织损伤较烫伤组明显减轻。结论:严重烫伤早期SD大鼠肠黏膜组织损伤明显,wnt5a可能参与了烧伤脓毒症的发生发展过程。胰岛素能减少其表达,可能是其保护肠黏膜机制的因素之一键词]烧伤;肠黏膜;Wnt5a;大鼠「中图分类号1R644「文献标志码]A[文章编号]1674-7887(2013)04-0287-04Expression of Wnt5a in intestinal mucosa of rats during the early stage after severe burninjury and the effect of the insulinQI Jun, HU Kesu, ZHANG Yun, ZHANG Yi(Department of Plastic Surgery, the Affiliated Hospital of Nantong Uniity, Nantong 226001)[Abstract] Objective: To investigate the changes of the expressions of Wnt5a in intestinal mucosa of rats during the earlyage after severe burn Injury and its sigficance. Methods: 48 healthy SD rats were set upscalded models, which wererandomly divided into control group, scald group and scald +insulin group(treatment group). The intestinal mucosa tissuesample separately after 6, 12, 24, 48 hours were posted scald injury. In the treatment group, subcutaneous injection of 3 U/kgsulin after scald injury. The expression of Wnt5a in the intestinal mucosa was detected with immunohistochemistry methodsnd analyzed by a micro-image analysis system, and at the same time we observed the pathological changes of the intestinalmucosa tissue of each groups. Results: In the scalded group, the expression of Wnt5a was higher than that in control group atthe same time(P<0.05),6-12 hours post scald injury the expression was increased. Following the expression of Wnt5adecreased obviously 24 hours post scald injury, 48 hours post scald injury the expression was increased. The expression ofWnt5a in the treatment group was significantly smaller than that in scalded group at most time points at the same time(P<0.05). Conclusions: In the early phase of severe burns injury, the intestinal mucosa tissue injury is very severe. Wnt5amay take part in the procession of burn sepsis. Insulin can deduce its expression, which may be one of factors protecting theIKey words burn; intestinal mucosa; Wnt5a;rat临床很早就发现严重的烧伤可导致胃、十二指量的细菌和内毒素,主要是依赖于其肠道屏障的功肠溃疡,甚至岀现消化道大岀血或穿孔等并发症。能。对其屏障的功能行深人地研究,有可能使烧伤的其影响因素有多种,目前尚未完全明了,胃肠黏膜缺救治水平再上1个台阶。Wnt5a为分泌性信号转导血和胃液氢离子反渗是主要的发病原因肠道是人体分子Wnt家族成员。近来,不少研究资料②显示内最大的“内毒素库”。正常的肠道之所以能抵抗大Wnt5a在单核巨噬细胞的激活中发挥着重要作用。*[基金项目]南通大学自然科学类科研课题(117017)*[作者简介]祁俊,男,汉族,生于1980年8月,江苏省南通市人,硕士,主治医师,研究方向:烧伤整形等*[通信作者]张逸,电话:0513-81168901,E- mail: qijunsky@126cm1005-1013for addiction[J]. Brain Res, 2011, 1406: 59-64[17 Kumar D, Deb L, Chakraborty J, et al. A polymorphism of收稿日期]2013-03-10the CreB binding protein(CrEBBP) gene is a risk fac