姜黄素对Aβ诱导的AD大鼠海马CRMP-2表达的影响

目的探讨姜黄素对Aβ诱导的老年痴呆大鼠认知功能和海马CRMP-2的影响。方法将SD大鼠随机分为空白对照组、AD对照组和姜黄素给药组。Aβ1-40微量注射至大鼠右侧海马制作AD大鼠模型。Morris水迷宫试验测定大鼠学习记忆能力。RT-PCR检测海马内CRMP-2mRNA的表达,Western blotting方法检测海马内CRMP-2和p-CRMP-2蛋白表达,免疫组织化学方法检测海马内轴突蛋白表达。结果姜黄素干预后AD大鼠空间学习记忆能力明显改善(P<0.05)。与空白对照组相比,AD对照组大鼠海马区CRMP-2表达显著降低(P<0.05),轴突蛋白NFP-200阳性纤维排列紊乱,不规则,纤维数目显著减少(P<0.05);而在姜黄素组大鼠海马区可见CRMP-2表达升高,p-CRMP-2表达降低,而轴突蛋白表达明显增加(P<0.01)。结论姜黄素能够改善Aβ1-40诱导的AD模型大鼠空间学习记忆障碍,其机制可能与姜黄素提高CRMP-2的表达并抑制CRMP-2的磷酸化从而促进轴突再生有关。     

miR-106a抑制胶质瘤细胞增殖并诱导凋亡

目的胶质瘤是最常见的原发性中枢神经系统肿瘤,具有较高的病死率。现有的研究表明miR-106a在多种肿瘤中表达上调,并发挥着致癌性作用,但miR-106a在胶质瘤中的表达和作用却并不清楚。方法不同级别胶质瘤组织和胶质瘤细胞系(T98G,SHG44,U87,U251,U373)内的miR-106a水平通过实时定量聚合酶链式反应(qRT-PCR)测定。转染miRNA寡聚核苷酸后的U87和U251细胞的增殖能力和细胞周期分别采用四甲基偶氮唑盐(MTT)比色法和流式细胞仪测定,并且通过膜联蛋白/碘化丙啶(annexin V/PI)双染法测定了miR-106a对胶质瘤细胞的凋亡影响。结果miR-106a在胶质瘤组织和胶质瘤细胞系内表达水平相对正常脑组织显著下降,并且miR-106a表达水平与胶质瘤病理级别负相关。胶质瘤细胞转染miR-106a后,可检测到过表达的miR-106a可显著抑制胶质瘤细胞的增殖能力,阻滞细胞周期于G0/G1期,同时诱导胶质瘤细胞凋亡增加。结论 miR-106a可阻滞胶质瘤细胞细胞周期、抑制增殖和诱导凋亡。     

乌司他丁对缺氧诱导PC12细胞神经损伤的保护作用研究

目的 探讨乌司他丁对缺氧诱导PC12细胞神经损伤的作用及分子机制。方法 PC12细胞分为对照组、缺氧组、缺氧+乌司他丁低、中、高剂量组、缺氧+miR-NC组、缺氧+miR-190组、缺氧+乌司他丁+anti-miR-NC组、缺氧+乌司他丁+anti-miR-190组。检测培养液中乳酸脱氢酶（LDH）漏出率及细胞中超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量；流式细胞术检测细胞凋亡；蛋白质印迹法（Western blotting）检测蛋白表达；实时荧光定量聚合酶链反应（qRT-PCR）检测miR-190表达水平。结果 与缺氧组比较，缺氧+乌司他丁低、中、高剂量组PC12细胞中LDH漏出率降低，SOD活性升高，MDA含量降低，细胞凋亡率降低，Bcl-2相对表达量升高，Bax相对表达量降低，miR-190相对表达量升高，且呈剂量依赖性（均P<0.05）。miR-190过表达后，LDH漏出率降低，SOD活性升高，MDA含量降低，细胞凋亡率降低，Bcl-2相对表达量升高，Bax相对表达量降低（均P<0.05）。抑制miR-190表达能逆转乌司他丁对缺氧诱导的PC12细胞损伤的作用。结论 乌司他丁可能通过上调miR-190表达对缺氧诱导PC12细胞神经损伤起保护作用。     

降低细胞胆固醇水平对β-淀粉样肽生成的影响

目的观察降低细胞胆固醇水平对β-淀粉样肽(β-amyloid,Aβ)生成的影响,初步探讨胆固醇和阿尔茨海默病(Alzheimer's disease,AD)的相关性。方法以稳定表达人野生型淀粉样前体蛋白(amyloid precursor protein,APP)的神经母细胞瘤SH-SY5Y细胞为模型,分别给予β-甲基环糊精或洛伐他汀对细胞系进行处理;胆固醇定量试剂盒测定细胞内胆固醇水平的变化,放射免疫法测定细胞培养液中Aβ的含量,Western Blot方法半定量检测全长型APP和可溶性APPα的水平。结果β-甲基环糊精和洛伐他汀处理组分别降低细胞胆固醇水平67.5%和49.5%(P<0.05),细胞培养液中Aβ含量分别降低39.5%和25.7%(P<0.05),sAPPα含量分别增加3.5倍和2.0倍(P<0.05)。结论降低细胞胆固醇水平使细胞外Aβ含量减少,sAPPα含量增加,这提示胆固醇可能通过影响APP代谢途径而参与AD的病理过程。     

miR-132靶向FoxO3a抑制细胞自噬在脑出血模型大鼠中的神经保护作用

目的研究微小RNA (miR)-132靶向叉形头转录因子O亚型3a (Fox03a)抑制细胞自噬在脑出血模型大鼠中的神经保护作用。方法成年雄性SD大鼠随机分为对照组、模型组、miR-阴性对照(NC)组、miR-132组,后三组采用Ⅶ型胶原酶注入苍白球的方式建立脑出血模型,对照组和模型组给予生理盐水侧脑室注射,miR-NC组和miR-132组分别给予miR-NC及miR-132侧脑室注射。造模后第7d,比较各组大鼠神经功能评分的差异,苏木精-伊红染色观察血肿周围脑组织病理改变,PCR检测miR-132 的表达水平,Western blot 检测 FoxO3a、自噬基因 (Beclin-1、Atg12、LC3) 的表达水平;培养 PC12 神经元细胞,转染miR-NC或miR-132后,采用双荧光素酶报告基因实验验证miR-132靶向FoxO3a。结果在动物实验中,模型组大鼠的神经功能评分增加,血肿周围脑组织中FoxO3a、Beclin-1、Atg12、LC3-Ⅱ/LC3-Ⅰ的表达增加,miR-132的表达减少(P0.05);miR-132组大鼠的神经功能评分降低,血肿周围脑组织中FoxO3a、Beclin-1、Atg12、LC3-Ⅱ/LC3-Ⅰ的表达减少,miR-132的表达增加(P0.05)。在细胞实验中,miR-132组细胞中FoxO3a的表达水平及野生型FoxO3a 3' UTR双荧光素酶报告基因的荧光活力均低于miRNC 组(P0.05)。结论 miR-132 在脑出血模型大鼠中起保护作用,靶向 FoxO3a 并抑制自噬是可能的分子机制。     

姜黄素对AD小鼠海马神经元凋亡和GSK-3β表达及磷酸化的影响

目的观察姜黄素对AD小鼠模型海马神经元凋亡和糖原合成酶激酶3β表达及其磷酸化影响。方法将20只APP/PS1转基因小鼠随机分为AD模型组、AD模型+姜黄素组,每组10只,AD模型+姜黄素组腹腔注射剂量为400mg/(kg·d)姜黄素,1次/d,连续14d。取12只正常小鼠作对照组。采用扫描电镜检测小鼠海马神经元凋亡,Western blot检测GSK-3β、酪氨酸磷酸化GSK-3β(pTyr-GSK-3β)和丝氨酸磷酸化GSK-3β(pSer-GSK-3β)的表达情况。结果与对照组相比,模型组海马神经元出现染色质边集、线粒体肿胀、细胞器减少;pTyr-GSK-3β和pSer-GSK-3β的表达明显增加(t=5.112,P=0.005;t=5.619,P=0.006)。与模型组相比,姜黄素组海马神经元染色质呈弥散分布、线粒体嵴清晰可见、细胞器排列紧密,pTyr-GSK-3β表达明显降低(t=-7.985,P=0.001),pSer-GSK-3β表达明显提高(t=9.105,P=0.001)。结论 GSK-3β可能参与AD小鼠海马神经元损伤,姜黄素通过减少pTyr-GSK-3β、增加pSer-GSK-3β的表达抑制GSK-3β活性来减少AD小鼠海马神经元凋亡。     

精神分裂症患者血清非编码小分子RNA-320a及RNA-320b表达的研究

目的:探讨精神分裂症患者血清非编码小分子RNA(miR)-320a、320b的表达。方法:应用实时荧光定量逆转录聚合酶链式反应技术(RT-PCR)检测30例首次发病的精神分裂症患者(发病组)、30例治疗后临床缓解的精神分裂症者(缓解组)和30名正常对照者(健康对照组)血清miR-320a及miR-320b表达水平。结果:发病组和缓解组血清miR-320a和miR-320b表达明显低于健康对照组(P均0.001),且发病组明显低于临床治愈组(P均0.05)。结论:精神分裂症患者血清miR-320b和miR-320a表达低,并与病情有关。     

姜黄素通过IGF-1/Akt/FoxO3a通路保护鱼藤酮诱导PC12细胞的PD模型

目的探究姜黄素(Cur)对鱼藤酮(Ro)诱导的PC12细胞损伤的保护作用及机制。方法建立鱼藤酮诱导的PC12细胞的帕金森病模型,利用姜黄素进行干预。实验分组为空白对照组,鱼藤酮模型组(终浓度:0.1μmol/L),姜黄素干预组(终浓度:0.5μmol/L、1.0μmol/L、5.0μmol/L、10μmol/L)。MTT比色法检测细胞活力,AO/EB荧光染色法观察细胞的凋亡情况,AnnexinⅤ-FITC/P双染检测细胞凋亡,Western-blot法检测细胞内IGF-1、Akt、FoxO3a的蛋白表达。结果 MTT结果显示:鱼藤酮组较对照组细胞活力明显降低,差异具有统计学意义(P<0.01),0.5μmol/L和1.0μmol/L姜黄素干预组可减轻0.1μmol/L鱼藤酮对PC12细胞增殖活力的影响,明显抑制了鱼藤酮对PC12细胞凋亡的诱导作用,与鱼藤酮组比较差异有统计学意义(P<0.01)Western-blot结果示:鱼藤酮组较对照组明显降低,差异具有统计学意义(P<0.01),0.5μmol/L和1.0μmol/L姜黄素干预组IGF-1、磷酸化的Akt(p-Akt)、磷酸化的FoxO3a(p-FoxO3a)表达与鱼藤酮组比明显升高,差异均有统计学意义(均P<0.01)。以上结果均显示5.0μmol/L和10μmol/L姜黄素干预组与鱼藤酮组比较差异无统计学意义(P>0.05);结论适宜浓度的姜黄素对鱼藤酮诱导PC12细胞损伤的PD模型具有保护作用,其保护作用呈浓度依赖性,其保护作用的机制可能与激活IGF-1/Akt/FoxO3a通路有关。     

姜黄素对多巴胺能细胞的保护作用研究

目的探讨姜黄素对多巴胺能细胞的保护作用及机制。方法采用神经毒素鱼藤酮(1μmol/L)处理经神经生长因子(NGF)(50 ng/ml×7)诱导分化过的PC12细胞,并在处理前6 h加入1μmol/L的姜黄素进行干预,MTT法检测细胞活力,荧光分光光度法检测细胞内活性氧水平,比色法检测还原型谷胱甘肽(GSH)含量。结果 NGF诱导后的PC12细胞经1μmol/L鱼藤酮处理24 h后,细胞活力下降至对照组的57.1%,细胞内活性氧水平为对照组的189%,而GSH水平显著下降(P<0.05);姜黄素干预后,与鱼藤酮组相比,细胞活力和GSH含量显著提高,活性氧水平显著降低(P<0.05)。结论姜黄素对多巴胺细胞具有保护作用,其机制与抗氧化活性有关。     

miR-20a/106b调控NKG2D途径介导儿童型脑干胶质瘤免疫逃逸的实验研究

目的探讨miR-20a/106b在儿童脑干胶质瘤(BSG)恶性进展中的作用及其机制。方法回顾性纳入2012年1月至2017年12月天津市环湖医院(天津市脑系科中心医院)神经外科接受手术切除的13例儿童BSG患者和5例成人BSG患者。采用免疫组织化学染色法检测肿瘤组织标本中自然杀伤细胞2家族成员D(NKG2D)配体MICA的表达情况,采用生物信息学方法筛选出调控MICA的miRNA。应用人脑胶质瘤LN229和U251细胞株通过荧光素酶报告基因方法验证筛选出的miRNA与MICA的靶向调控关系,通过蛋白质免疫印迹(WB)实验检测miR-20a/106b转染LN229和U251细胞株后MICA的表达情况,通过乳酸脱氢酶(LDH)释放法检测免疫细胞对LN229细胞株的毒性能力。通过向去胸腺幼年(3周龄)和成年(10周龄)雄性SD大鼠脑桥区立体定向注射LN229细胞株的方法制备BSG幼鼠(J-rat)和成年鼠(A-rat)模型,并根据注射细胞株不同各分为3组,依次为J-rat组、J-rat-scr组、J-rat-miR-20a/106b-d组和A-rat组、A-rat-scr组、A-rat-miR-20a/106b-u组(每组各3只),通过免疫组织化学染色方法检测MICA的表达情况,采用MCID软件检测肿瘤的侵袭性,采用TUNEL法检测细胞凋亡。结果成人患者的MICA总阳性表达率较儿童患者高(分别为5/5、10/13,P<0.05)。检索结果显示,有23个可能调控MICA的microRNA,其中包括miR-20a和miR-106b。在LN229细胞株中,荧光素酶报告基因结果显示,转染pGL3-MICA-wt(野生型)质粒的荧光强度较对照组和转染pGL3-MICA-mut(突变型)质粒的强(均P<0.05);WB结果显示,与空白对照组和无义序列组比较,AS-miR-20a组、AS-miR-106b组及AS-miR-20a/106b组MICA的表达均增加(均P<0.05)。上述实验在U251细胞株得到相似的结果。不同效靶比(20∶1,10∶1,5∶1)情况下,AS-miR-20a/106b组LDH的活性高于对照组和AS-miR-20a/106b+MICA mAb组(均P<0.05),但后两组的差异无统计学意义(均P>0.05)。术后头颅MRI结果显示,3组幼鼠和3组成年鼠均成功制备脑干胶质瘤模型。MCID软件检测结果显示,J-rat-miR-20a/106b-d组I值较J-rat组、J-rat-scr组均降低(均P<0.05);A-rat-miR-20a/106b-u组I值较A-rat组、A-rat-scr组均增加(均P<0.05)。免疫组织化学染色结果显示,J-rat-miR-20a/106b-d组的MICA染色强度均较J-rat组和J-rat-scr组增加(均P<0.05),A-rat-miR-20a/106b-u组MICA的染色强度均较A-rat组和A-rat-scr组降低(均P<0.05)。体内凋亡实验显示,J-rat-miR-20a/106b-d组的细胞凋亡指数均较J-rat组和J-rat-scr组增加(均P<0.05),A-rat-miR-20a/106b-u组的细胞凋亡指数较A-rat组和A-rat-scr组均降低(均P<0.05)。结论miR-20a/106b通过NKG2D途经达到的免疫逃逸是导致儿童型BSG恶性度高的重要原因。     

Continuous stress disrupts immunostimulatory effects of IL-12

Immune stimulation by biological response modifiers is a common approach in tumor immunotherapy. IL-12 was found effective in various animal studies, but clinical trials showed limited success. However, among other differences, animal models do not simulate psychological or physiological stress while employing IL-12, whereas cancer patients often experience distress while treated with immunostimulants. Thus, in the current study we assessed the impact of continuous stress on the efficacy of IL-12 immunostimulation. F344 rats were subjected to a pharmacological stress paradigm (continuous administration of a β-adrenergic agonist) or to a 20 h behavioral stress paradigm (wet cage exposure) commencing 2 h before IL-12 administration. Twenty-six hours after stress initiation, we studied indices known to reflect IL-12 immunostimulatory impacts, including NK cell numbers and activity in different immune compartments, and in vivo resistance to MADB106 lung tumor colonization. The results indicated that both the pharmacological and behavioral stress paradigms significantly reduced the increase in the number and activity of marginating-pulmonary NK cells evident in non-stressed IL-12 treated animals. Additionally, stressed animals exhibited a lower IL-12-induced improvement of MADB106 lung clearance, an in vivo index that markedly depends on total marginating-pulmonary NK activity. These deleterious effects of stress were more prominent in males than in females. Overall, the findings demonstrate that prolonged stress exposure can disrupt the efficacy of simultaneous immunostimulatory treatments, irrespective of stress effects on baseline immune measures. Neuroendocrine and cellular mediating mechanisms are yet unknown, but the potential clinical ramifications of these findings warrant consideration in clinical trials employing immunostimulatory agents.     

PrP106—126对原代大脑皮质神经元的毒性作用

目的:探讨PrP10 6- 12 6对神经元的毒性作用。方法:原代培养的皮质神经元暴露于合成多肽PrP10 6 -12 6,观察细胞存活率的变化,采用流式细胞术检测有无凋亡的发生,用免疫印迹法检测caspase 3的活性。结果:在PrP10 6 -12 6的作用下,神经元存活数较对照组减少,随着作用时间的延长,存活率明显下降。在PrP10 6- 12 6作用下神经元的凋亡比例明显增加,PrP10 6- 12 6组凋亡细胞为2 4.93 % ,对照组为6.95 %。与对照组比,PrP10 6 -12 6组在培养第6、8、10天时均有较明显的caspase- 3表达,且伴有caspase 3的活化。结论:PrP10 6- 12 6对原代培养的皮质神经元有毒性作用,呈时间依赖性。PrP10 6 -12 6毒性作用涉及了细胞凋亡机制,激活caspase -3诱导细胞凋亡可能是途径之一。     

Intracerebral interleukin-1β impairs response to tumor invasion: involvement of adrenal catecholamines

Interleukin-1β (IL-1β) is released within the brain following stress, trauma, infection, and in specific brain disorders. This centrally acting IL-1β has recently been shown to impair peripheral immunity. Central administration of IL-1β suppresses natural killer (NK) cell activity impairs lung clearance of tumor cells and enhances tumor colonization. Using an in vivo model of tumor colonization (lung clearance of NK-sensitive MADB106 adenocarcinoma cells), this study examined the role of the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS) in mediating these effects. We demonstrate that adrenalectomy significantly attenuated the impaired lung clearance of MADB106 tumor cells induced by intracerebroventricular (i.c.v.) administration of IL-1β (20 ng). Supplementing adrenalectomized animals with corticosterone did not reinstate the effect. The effect of IL-1β on lung clearance was blocked by pretreatment with the β-adrenergic antagonist, nadolol (0.5 mg/kg), but not by the α-antagonist phentolamine (5 mg/kg). Peripheral noradrenergic pathways are not implicated given that systemic administration of the noradrenergic neurotoxin, 6-hydroxydopamine, did not block the effect of IL-1β. Taken together, these findings indicate that IL-1β impairs lung clearance of MADB106 tumor cells via the actions of adrenal catecholamines, most likely epinephrine, acting at β-adrenergic receptors in the periphery.     

PrP 106—126肽段对分化PC12细胞能量代谢的影响

目的:研究朊蛋白106-126肽段对分化PC12细胞能量代谢的影响。方法:PC12细胞培养后加入神经生长因子(NGF),再加入朊蛋白106-126肽段,观察细胞存活率、线粒体形态学、线粒体膜电位和Ca2 -ATPase活性的改变。结果:细胞接触肽段后存活率下降28.9%;光镜下可见细胞贴壁不良,胞体缩小,细胞突起断裂缩短;电镜下可见核移位,染色质凝集,部分胞核呈碎块状,线粒体肿胀、嵴紊乱、断裂;膜电位和Ca2 -ATPase的活性随着肽段的作用时间的延长而减小。结论:朊蛋白106-126肽段对分化PC12细胞具有毒性作用,能量代谢障碍可能是其毒性作用机制之一。     

Forelimb dyskinesia mediated by high‐frequency stimulation of the subthalamic nucleus is linked to rapid activation of the NR2B subunit of N‐methyl‐d‐aspartate receptors

Dyskinesia is a major side‐effect of chronic l ‐DOPA administration, the reference treatment for Parkinson’s disease. High‐frequency stimulation of the subthalamic nucleus (STN‐HFS) alleviates parkinsonian motor symptoms and indirectly improves dyskinesia by decreasing the l ‐DOPA requirement. However, inappropriate stimulation can also trigger dyskinetic movements, in both human and rodents. We investigated whether STN‐HFS‐evoked forelimb dyskinesia involved changes in glutamatergic neurotransmission as previously reported for l ‐DOPA‐induced dyskinesias, focusing on the role of NR2B‐containing N‐methyl‐d ‐aspartate receptors (NR2B/NMDARs). We applied STN‐HFS in normal rats at intensities above and below the threshold for triggering forelimb dyskinesia. Dyskinesiogenic STN‐HFS induced the activation of NR2B (as assessed by immunodetection of the phosphorylated residue Tyr¹⁴⁷²) in neurons of the subthalamic nucleus, entopeduncular nucleus, motor thalamus and forelimb motor cortex. The severity of STN‐HFS‐induced forelimb dyskinesia was decreased in a dose‐dependent manner by systemic injections of CP‐101,606, a selective blocker of NR2B/NMDARs, but was either unaffected or increased by the non‐selective N‐methyl‐d ‐aspartate receptor antagonist, MK‐801.     

Intracerebral HIV glycoprotein (gp120) enhances tumor metastasis via centrally released interleukin-1

Infection with the human immunodeficiency virus (HIV) is associated with a high incidence of cancers. This relationship does not appear to be due to a direct effect of the virus, and may be mediated by neuroimmune interactions since the HIV glycoprotein, gp120, enters the brain soon after infection with HIV, and intracerebroventricular (i.c.v.) infusion of gp120 suppresses aspects of cellular and tumor immunity. It has been speculated that this suppression may be attributed to the release of interleukin-1 (IL-1) in the brain induced by gp120. Using an in vivo tumor model, we examined the effect of centrally administered gp120 on tumor metastasis and lung clearance of mammary adenocarcinoma (MADB106) tumor cells in rats, and the role played by brain IL-1 in mediating these effects. We demonstrate that central administration of gp120 (4 μg) significantly (p<0.05) increased the retention of tumor cells in the lungs and significantly (p<0.02) enhanced the development of tumor metastases. Central administration of IL-1β (10 ng) also significantly (p<0.05) increased retention of tumor cells in the lungs. The effect of gp120 on lung retention of tumor cells was blocked by co-administration of α-melanocyte stimulating hormone (α-MSH, 20 ng), a hormone that blocks many of the biological effects of IL-1, or the IL-1 receptor antagonist (50 μg). Given that systemic administration of gp120 or IL-1β had no effect on the retention of tumor cells in the lungs, these findings indicate that gp120-induced secretion of IL-1 within the brain most likely mediates the effects of gp120 on tumor metastasis. These findings suggest a possible neuroimmune mechanism to account for the increased incidence and aggressiveness of tumors in HIV-infected patients.     

Study on the toxic mechanism of prion protein peptide 106-126 in neuronal and non neuronal cells

A synthetic peptide corresponding to the 106-126 amyloidogenic region of the cellular human prion protein (PrP(c)) is useful for in vitro study of prion-induced neuronal cell death. The aim of the present work was to examine the implication of the cellular prion protein in the toxicity mechanism induced by PrP 106-126. The effect of PrP 106-126 was investigated both on human neuroblastoma SH-SY5Y cells and on SH-SY5Y overexpressing murine cellular prions (wtPrP). We show by metabolic assay tests and ATP assays that PrP(c) expression does not modulate the toxicity of the prion peptide. Moreover, we investigated the effect of this peptide on an established non neuronal model, rabbit kidney epithelial A74 cells that express a doxycycline-inducible murine PrP(c) gene. We show for the first time that the prion peptide 106-126 does not exert any toxic effect on this cell line in the presence or absence of doxycycline. Our results show that the PrP 106-126-induced cell alteration is independent of PrP(c) expression. Rather, it seems to act via an interaction with lipidic components of the plasma membrane as strengthened by our results showing the differential susceptibility of neuronal and non neuronal cell lines that significantly differ by their membrane fatty acid composition.