【作者】 林焕冰；

【导师】 徐江平； 李铁军；

【作者基本信息】 南方医科大学 ， 药理学， 2010， 博士

【摘要】 背景：阿尔茨海默病(Alzheimer’s disease, AD)是一种发生于老年期或老年前期的中枢神经系统退行性疾病。AD典型的病理特征为：神经元内纤维缠结(Neufibrillary tangles, NFTs)、细胞外老年斑(Senile plaques, SP)的形成,以及中枢胆碱能神经元的大量死亡与丢失。β淀粉样蛋白(amyloidβ, Aβ)被认为是AD病患者脑组织SP的最主要组成成分,它由β分泌酶和γ分泌酶裂解淀粉样蛋白前体蛋白(amyloid precursor protein, APP)产生。目前,AD发病机制还不是很清楚,但有研究显示β淀粉样蛋白(amyloidβ, Aβ)是各种因素诱发AD的共同通路,是AD形成和发展的关键因素。C反应蛋白(C reactive protein, CRP)是由炎症或组织损伤引起的非特异的急性时相反应中最具代表性的标志物。目前越来越多的研究表明CRP可能参与AD的发病机制,并能恶化认知功能。但仍未有直接的证据证明CRP能导致AD认知障碍,并引起神经化学物质变化。目的：为探讨CRP对学习记忆及AD发病机制相关分子的影响,通过研究大鼠认知行为的影响及海马和皮层中Aβ产生增多相关的分子(APP、PS-1、PS-2及BACE)和炎症因子(CRP、IL-1β、IL-6及TNF-α)蛋白水平和基因水平的变化,以明确CRP能否介导大鼠学习记忆障碍及其可能机制。继而从细胞水平进一步探索其可能的作用机制。采用PC12细胞为研究对象,系统地研究CRP对PC12细胞毒性作用及可能机制,为CRP成为治疗AD的新靶点提供理论依据。方法：(1)40只雄性Sprague-Dawley大鼠(220 g～250 g)随机分为正常对照组、假手术组、CRP组和Aβ25-35组。采用双侧脑室立体定位(i.c.v.)注射给药,CRP组和Aβ25-35组分别相应给予CRP(25.6μg/只)和Aβ25-35(10μg/只),假手术组给予等体积空白溶剂；手术14天后,采用Morris水迷宫进行定向航行实验及空间探索实验检测其参照和空间学习记忆能力；在术后第20天采用避暗法检测其长期记忆能力；行为学检测后,采用实时定量逆转录多聚酶链反应(Real-time RT-PCR)和免疫印迹(Western blotting)方法检测海马和皮层组织中与“Aβ假说”分子机制相关分子(APP、PS-1、PS-2及BACE)及“神经炎症假说”分子机制相关炎症因子(CRP、IL-1β、IL-6及TNF-α)的基因和蛋白表达水平变化。(2)CRP的细胞毒性实验：体外培养PC12细胞,应用四甲基偶氮唑蓝法(methyl thiazolyl tetrazolium, MTT)观察不同浓度CRP对细胞活力的影响,并测定细胞外液乳酸脱氢酶(Lactate dehydrogenase, LDH)活力。(3)采用Real-time PCR和酶联免疫吸附法(ELISA)研究在亚毒性浓度下作用48小时,CRP浓度作用(1.25mg/L、2.5mg/L和5 mg/L)对Aβ生成增加相关基因(PS-1, PS-2, BACE-1和APP) mRNA表达和Aβ1-42表达的影响。(4)采用Real-time PCR和酶联免疫吸附法(ELISA)研究在亚毒性浓度下(5mg/L),CRP刺激后不同时间(12小时、24小时和48小时),PC12对Ap生成增加相关基因(PS-1, PS-2, BACE-1和APP) mRNA表达和Aβ1-42表达的影响。结果：(1)在Morris水迷宫定向航行实验中,游泳轨迹结果表明尽管在训练初期大多数动物都是沿边搜索方式,但正常组和假手术组比CRP组和Aβ组更快地学会直线搜索方式,导致显著缩短潜伏期和游泳路程。实验结果表明,在Day 1各组动物寻找平台的潜伏期和所需游泳路程无显著性差异(F=1.062,P=0.379和F=0.426,P=0.736)；其余四天各组动物寻找平台的潜伏期有显著性差异(分别为F=11.257,P=0.000；F=3.7、49,P=0.021；F=5.569,P=0.003和F=6.359,P=0.002),寻找平台的所需游泳路程有显著性差异(分别为F=7.658,P=0.001；F=5.925,P=0.002；F=5.421,P=0.004和F=5.780,P=0.003)。CRP组大鼠与假手术组大鼠比较,在Day 2、Day 4和Day 5寻找平台的潜伏期有显著性延长(分别为P=0.001；P=0.020和P=0.006),在Day 2、Day 3、Day 4和Day 5寻找平台的所需游泳路程有显著性延长(分别为P=0.001；P=0.039；P=0.027和P=0.035)；Aβ25-35组大鼠与假手术组大鼠比较,在Day 2、Day3、Day 4和Day 5寻找平台的潜伏期有显著性延长(分别为P=0.000；P=0.034；P=0.011和P=0.006),在Day 2、Day 3、Day 4和Day 5寻找平台的所需游泳路程有显著性延长(分别为P=0.001；P=0.002;P=0.011和P=0.026)；但正常组与假手术组相比无显著性差异。空间探索实验中,各组大鼠90秒内游泳的总路程无显著性差异(F=0.157,P=0.925),但各组大鼠90秒内停留在原平台所在象限(即目的象限)的探索时间和探索路程有显著性差异(F=5.405,P=0.004；F=3.221,P=0.036)。与假手术组相比,CRP组和Ap组大鼠90秒内停留在目的象限的探索时间明显缩短(分别为P=0.048和P=0.019)；与假手术组相比, CRP组和Aβ25-35组大鼠90秒内在目的象限探索路程明显缩短(分别为P=0.044和P=0.032)；但正常组与假手术组相比均无显著性差异。提示CRP组和Aβ25-35组大鼠的空间记忆能力损伤。在被动避暗实验的适应训练中,各组大鼠进入暗室的潜伏期无显著性差异(F=0.115,P=0.951),表明各组大鼠间喜暗及钻洞的习性无显著性差异。在测试实验中,各组大鼠进入暗室的潜伏期均有显著性差异(F=6.870,P=0.001)。与假手术组相比,CRP组和Aβ25-35组大鼠均进入暗室的潜伏期明显缩短(分别为P=0.002和P=0.025)；但正常组与假手术组相比均无显著性差异。表明CRP组和Aβ25-35组大鼠的长期记忆受损。大鼠双侧i.c.v.注射后第22天,Real-time PCR结果显示，与假手术组相比,CRP组能显著提高在皮层组织中APP(P=0.031),IL-1β(P=0.000),IL-6(P=0.048),TNF-α(P=0,000),PS-1(P=0.035),PS-2(P=0.025)和内源性CRP(P=0.002)mRNA表达和提高海马组织中APP(P=0.019),IL-1β(P=0.000),IL-6(P=0.014),TNF-α(P=0.007)和内源性CRP(P=0.002)mRNA表达；Ap组也能显著提高在皮层组织中APP(P=0.013),IL-1β(P=0.000),IL-6(P=0.002),TNF-α(P=0.000),PS-1(P=0.033),PS-2(P=0.020)和内源性CRP(P=0.000)mRNA表达和提高海马组织中APP(P=0.004),IL-1β(P=0.000),IL-6(P=0.017),TNF-α(P=0.002)和内源性CRP(P=0.035)mRNA表达。但正常组与假手术组相比均无显著性差异。Western blotting结果表明,与假手术组相比,CRP组能显著提高在皮层组织中APP(P=0.001),IL-1β(P=0.003)和IL-6(P=0.006)蛋白表达和提高海马组织中IL-1β(P=0.000),BACE(P=0.000),TNF-α(P=0.000)和APP(P=0.000)蛋白表达；Aβ组也能显著提高在皮层组织中APP(P=0.002),IL-1β(P=0.020)和IL-6(P=0.024)蛋白表达和提高海马组织中IL-1β(P=0.000),BACE (P=0.000),TNF-α(P=0.000)和APP(P=0.000)蛋白表达。但正常组与假手术组相比无显著性差异。(2)细胞实验结果表明CRP对PC12细胞产生毒性作用,细胞存活率下降,LDH外漏增加,并且呈一定的剂量依赖性；当CRP浓度在12.5 mg/L、25 mg/L、50 mg/L和100 mg/L作用48小时后,与正常对照组比较,细胞存活率显著下降(分别为P=0.000,P=0.001,P=0.003和P=0.003)；细胞LDH外漏显著增加(分别为P=0.001,P=0.007,P=0.032和P=0.042)。(3)在亚毒性浓度下,CRP呈剂量依赖性地提高PS-1, PS-2, BACE-1和APP mRNA表达和Aβ1-42表达。不同浓度CRP (1.25 mg/L、2.5 mg/L和5 mg/L)能提高PC12细胞对PS-1, PS-2, BACE-1和APP mRNA表达及显著提高Aβ1-42表达(分别为P=0.001,P=0.000和P=0.000),且呈一定的剂量依赖性；当CRP的浓度为5 mg/L时,PC12细胞对PS-1, PS-2, BACE-1和APP mRNA表达显著上升(分别为P=0.000,P=0.000,P=0.000和P=0.000)。(4)CRP呈时间依赖性地提高PS-1, PS-2, BACE-1和APP mRNA表达和Aβ1-42表达。在亚毒性浓度(5 mg/L)下,CRP刺激后不同时间(12小时、24小时和48小时),PC12细胞PS-1, PS-2, BACE-1和APPmRNA表达升高,并显著提高Aβ1-42表达(分别为P=0.026和P=0.001),且呈一定的时间依赖性；当5 mg/L CRP刺激48小时后,PC12细胞对PS-1, PS-2, BACE-1和APP mRNA表达显著上升(分别为P=0.000,P=0.003,P=0.000和P=0.003),且BACE-1 mRNA表达在刺激24小时后也显著上升(P=0.009)。结论：i.c.v.注射CRP后能导致大鼠学习记忆障碍,推测其作用机制：一方面,可能通过上调APP, BACE和PS,进而导致内源性的Aβ大量生成；另一方面,可能通过诱导神经炎症反应发生,从而导致一系列病理损伤,最终导致认知功能障碍；CRP对PC12细胞能产生明显细胞毒性作用,且呈浓度依赖性,其作用机制通过上调APP, PS-1, PS-2和BACE-1的mRNA表达,进而大量生成内源性的Ap,最终产生细胞毒性。CRP可能成为治疗AD的一个新靶点。更多还原

【Abstract】 Background:Alzheimer’s disease (AD) is a progressively neurodegenerative disorder in older. Its pathologycal features include intracellular neurofibrillary tangles(NFT), and senile plaques (SP), which primarily consist of extracellularβ-amyloid peptides (Aβ). Aβpeptides are generated by sequential cleavages of APP byβ-andγ-secretases. Although the pathogenesis of AD is unclear, Ap is considered to be a common factor and key link of AD. C-reactive protein (CRP), a prototypic acute-phase protein, is a sensitive marker of inflammation and tissue damage. There is much evidence showing that CRP may be implicated in the pathogenesis of AD and contribute to the cognitive problem in AD. However, there is no direct evidence showing that CRP impairs memory and causes neurochemical changes as those observed in AD.Objective:To investigate the effect of CRP, associated with Alzheimer’s disease (AD), on cognitive deficits and molecular biologial alteration in rat, we examined the effects of CRP on memory performance and levels of inflammatory cytokines (IL-1βp, IL-6, and TNF-α), CRP, and markers of the endogenous production of Aβ(APP, PS-1, PS-2 and BACE) in rats, which are associated with AD. In addition, in order to further study the mechanism of CRP impair momory, we determined the cytotoxicity of CRP and the concentration and time effects of CRP using PC12 cells.Methods:(1) Forty Male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into four groups with ten animals each:control (naive animals) group, vehicle group, CRP group, and Aβ25-35 group (positive control). Animals were infused with same voleme of sterile distilled water (vehicle), aggregated Aβ25～35 (10μg/side), or CRP (12.8μg/side) into each cerebral lateral ventricle. Two weeks after operation, the place navigation test and spatial probe test were performed to evaluate both reference and spatial memory in the Morris water-maze. From the 20th to the 21th day, long-term memory was measured by using the passive avoidance test. The mRNA levels of inflammatory cytokines (IL-1β, IL-6, TNF-α), endogenous CRP, APP, PS-1and PS-2 in the hippocampus and cerebral cortex were measured by real time RT-PCR. The protein levels of inflammatory cytokines (IL-1β, IL-6, TNF-α), endogenous APP and BACE in the hippocampus and cerebral cortex were measured by Western blot. (2) In order to investigate the cytotoxicity of CRP, the methyl thiazolyl tetrazolium (MTT) assay was applied to evaluate the cell viability and biochemical method was used to determine the lactate dehydrogenase (LDH) activity in PC 12 cells. (3) In order to investigate the concentration effects of CRP, the mRNA expression levels of PS-1, PS-2, BACE-1 and APP were determined by Real-time PCR and ELISA was applied to evaluate the content of Aβ1-42. (4) In order to investigate the time effects of CRP, the mRNA expression levels of PS-1, PS-2, BACE-1 and APP were determined by Real-time PCR and ELISA was applied to evaluate the content of Aβ1-42.RESULTS:(1) In the place navigation test, behavioral tracking results revealed that, although most rats showed surrounding searches at the beginning of training, the naive and vehicle-treated rats learned more quickly than the drug-treated rats to swim away from the side walls to find the platform in the target quadrant, leading to shorter escape latency and swimming path length. The latency to reach the platform and the swimming distances during the first training trial were not changed in rats treated with Aβ25-35 or CRP, compared to the vehicle controls. During days 2-5, Aβ25-35-or CRP-treated rats displayed increases in the latency to reach the platform and the swimming distances, relative to vehicle-treated or naive rats (P<0.01, P<0.05.) However, these were no significance between vehicle-treated and naive rats(P>0.05). In the spatial probe trial, the distances traveled in all the four quadrants were not different among groups. Compared to the vehicle control, Aβ25-35 decreased both the duration and swimming distance (P<0.05, P<0.01) in the target quadrant. Similarly, CRP also decreased both indices (P<0.01 for duration and P<0.05 for distance) relative to the vehicle control, suggesting impairment of spatial memory. In addition, all the rats took approximately 23 s before entering the dark compartment, regardless of the treatment during the training; there was no difference among groups (P>0.05). In contrast,24 h after initial training (21d post-infusion), both naive and vehicle-treated rats displayed significantly increases in retention, suggesting that animals remembered the association of the aversive stimulus with the dark compartment. In contrast, the retention was significantly decreased in rats treated with either Aβ25-35 or CRP (P<0.01 and P<0.001, respectively), compared to the vehicle control, suggesting impaired long-term memory. Real-time RT-PCR revealed that both CRP and Aβ25-35 increased the mRNA levels of APP, IL-1β, IL-6, TNF-α, and CRP in the cerebral cortex and hippocampus. Immunoblotting analysis revealed that the protein levels of total APP, IL-1β, and IL-6 were deferentially changed by CRP in the cerebral cortex and hippocampus. The expression of APP, IL-1β, and IL-6 was increased, but that of BACE and TNF-αwas unaltered in the cerebral cortex; in the hippocampus, the expression of all the five proteins except IL-6 was increased. (2) In the cytotoxicity test, the results demonstrated that the concentration-dependent effects of CRP on the viability of PC 12 cells as well as on the LDH leakage of PC 12 cells. The viability significantly decreased and the LDH leakage significantly increased when the PC12 cells treated with CRP at the concentration between 12.5 and 100mg/L for 48h (P<0.01, P<0.05). (3) The concentration-dependent upregulated effects of CRP on the mRNA expression levels of PS-1, PS-2, BACE-1 and APP as well as on the content of Aβ1-42. The content of Aβ1-42 and the mRNA expression levels of PS-1, PS-2, BACE-1 and APP increased significantly while PC12 cells treated with CRP at the subtoxic concentration of 5 mg/L for 48h (P<0.01, P<0.05). (4) The time-dependent upregulated effects of CRP on the mRNA expression levels of PS-1, PS-2, BACE-1 and APP as well as on the content of Aβ1-42. The content of 2 and the mRNA expression levels of PS-1, PS-2, BACE-1 and APP increased significantly while PC 12 cells treated with CRP at the subtoxic concentration of 5 mg/L for 48 h (P<0.01, P<0.05). Particularly, the mRNA expression levels of BACE-1 also significantly increased for 24h (P<0.01)CONCLUSION:CRP contributes to memory loss and early phase of pathogenesis of AD. CRP can be a novel target for therapeutic intervention in AD, in particular in the memory loss associated with AD.更多还原