Alzheimer病星形胶质细胞和海马神经元胰岛素信号通路蛋白变化的研究
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摘要
研究背景
阿尔茨海默病(Alzheimer's disease, AD)是一种神经退行性疾病,也是老年人痴呆中最常见的类型,表现为进行性加重的认知功能障碍和神经精神症状及生活能力的逐渐丧失。该病发病率高,是继心脑血管疾病和肿瘤之后导致人类死亡的主要原因,正日益成为对老年人健康的重要威胁。AD是一种多因素共同作用而致的复杂异质性疾病,淀粉样蛋白(beta-amyloid protein, Aβ)沉积、神经存活通路紊乱、糖代谢障碍、线粒体损伤、氧化应激和炎症等均参与疾病的发生发展。其中,Ap沉积是公认的“源头”,尤其以Aβ1-42寡聚体毒性最强,而其他的病理改变和致病途径可能是Aβ过量沉积的继发结果。
胰岛素信号通路参与能量代谢和神经内分泌两个过程,能调节糖脂代谢、突触形成及重塑、细胞存活和生长、炎症反应、线粒体功能和学习记忆等。该通路破坏不仅与糖尿病和肥胖等疾病相关,而且与神经退行性变及认知功能下降密不可分,且已经证实该通路损伤后能增加痴呆的发病风险。哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)是蛋白激酶B(protein kinase B, Akt/PKB)的下游底物之一,并且能调节星形胶质细胞谷氨酸转运体的表达。两者还能通过相互作用来调节突触活动及记忆形成。因此,增强胰岛素信号通路的活性能起到神经保护及改善认知的作用。
星形胶质细胞是脑组织的支持细胞,为神经元提供稳定的内环境。具体而言,星形胶质细胞能维持突触的正常功能、转移和储藏信息、参与认知、产生营养因子、移除毒素和代谢产物、维持正常的氧化还原电势以及调节神经递质和离子的浓度等。最重要的是,星形胶质细胞表达有大量胰岛素受体(insulin receptor, IR),并能受胰岛素的影响。谷氨酸是中枢神经系统(central nervous system, CNS)中最重要的兴奋性神经递质,在学习和记忆中起举足轻重的作用。谷氨酸在神经元活动时释放到突触间隙中,然后被星形胶质细胞上的兴奋性氨基酸转运体(excitatory amino acid transporters, EAATs)快速重吸收。该转运体被破坏的直接后果是导致谷氨酸在细胞外过度蓄积而对神经元产生兴奋性毒性损伤,而这也是多种神经退行性疾病包括AD的发病机制之一
研究目的
观察Aβ1-42寡聚体对人星形胶质细胞的影响,探索人星形胶质细胞上是否存在insul in/Akt/EAAT通路,以及Aβ1-42寡聚体和胰岛素对该通路蛋白表达水平和活性的影响作用；同时还观察Aβ1-42寡聚体侧脑室灌注AD模型大鼠的行为学改变及海马神经元胰岛素信号转导通路相关蛋白表达的变化；进一步寻找AD的发病机制及有效的干预靶点。
研究方法
1.Aβ1-42寡聚体的制备和鉴定。我们分别使用基础的方法和改良的方法制备Aβ1-42寡聚体,后者能在短时期内相对稳定保存。具体而言,首先,将Aβ1-42冻干粉与室温平衡后加入六氟异丙醇(hexafluoro-isopropanol, HFIP)使其单体化,再用无水二甲基亚砜(dimethyl sulfoxide, DMSO)溶解Aβ1-42肽膜。然后,使用SDS-PBS重悬Ap-DMSO溶液并于4℃孵育24h。最后,用PBS稀释重悬液,再于4℃孵育2周后用不含血清的DMEM稀释至实验所需的浓度。Ap1-42寡聚体制备完成后在透射电镜下进行观察和鉴定。
2.细胞分组和药物干预。星形胶质细胞共分为6组,待饥饿处理结束后即加药。C组为空白对照组,即给予无胎牛血清(fetal bovine serum, FBS)的DMEM;I组给予100nmol/L的Aβ1-42寡聚体溶液；II组给予1μmol/L的Aβ1-42寡聚体溶液。将三组细胞置于37℃、饱和湿度、5%CO2含量的恒温细胞培养箱中培养24h。后三组是在前三组的基础上均再给予100nmol/L人合成胰岛素作用30min,即III组为给予无FBS的DMEM培养24h后再给予100nmol/L胰岛素作用30min;Ⅳ组为给予100nmol/L的Aβ1-42寡聚体溶液培养24h后再给予100nmol/L胰岛素作用30min;Ⅴ组为给予1μmol/L的Aβ1-42寡聚体溶液培养24h后再给予100nmol/L胰岛素作用30mmin。
3.观察星形胶质细胞对药物的反应及检测insulin/Akt/EAAT通路相关蛋白的表达和活性。首先,用荧光定量RT-PCR和Western blot检测胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)的mRNA和蛋白水平,再通过免疫荧光染色的方法观察细胞形态变化。然后,使用荧光定量RT-PCR检测IR, Akt、 mTOR、EAAT1和EAAT2的mRNA水平；使用Western blot的方法检测IR-α IR-β、磷酸化胰岛素受体(phosphorylation of insulin receptor, p-IR, Y1361)、Akt、磷酸化蛋白激酶B (phosphorylation of protein kinase B, p-Akt, S473) mTOR、磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylation of mammalian target of rapamycin, p-mTOR, S2448), EAAT1和EAAT2的蛋白水平；使用免疫荧光染色的方法进一步观察IR-α、IR-β口p-IR在细胞内的表达情况。
4.星形胶质细胞活性的检测。使用MTT的实验方法检测星形胶质细胞在Aβ1-42寡聚体和胰岛素作用下细胞活性的变化。
5.动物分组和AD动物模型的建立。3-4月龄的健康成年雄性Wistar大鼠50只,体重为225±25g。术前行为学检测淘汰5只,将剩余45只大鼠分为3组。即正常对照组(C组,大鼠仅行手术而未行任何注射)、生理盐水组(NS组,向大鼠侧脑室内缓慢持续泵入生理盐水)和Aβ1-42寡聚体注射组(AD组,向大鼠侧脑室内缓慢持续泵入Aβ1-42寡聚体),各组动物数量均为15只(n=15)。先将Aβ1-42寡聚体溶液或者生理盐水注满微渗泵后再把各部分装置组装起来,并在脑立体定位仪的作用下植入微渗泵。调节流量调节器,使Aβ1-42寡聚体溶液或者生理盐水缓慢持续泵入,每天泵入3μL,连续给药30天。所有操作均遵循无菌操作的原则。术后待大鼠清醒后进行神经功能评定,并常规给予肌肉注射青霉素8万单位以防止切口感染,连续给药3天。
6.行为学检测。给药结束后行Morris水迷宫实验评估大鼠的学习和记忆能力。术后第31-34天进行定向航行实验,第35天进行空间探索实验。将平台置于南象限(随机预设),记录分析每只大鼠的逃避潜伏期,即在定向航行实验时大鼠到达平台的时间；记录分析空间探索实验时大鼠经过平台原位置的次数；记录分析空间探索实验时大鼠在南象限停留的时间；记录分析空间探索实验时大鼠在南象限游动的路程；计算空间探索实验时大鼠在南象限游动的时间及路程占总时间和总路程的比率。
7.大鼠海马神经元胰岛素信号通路相关蛋白表达的检测。麻醉大鼠,取材、固定海马组织后行免疫组织化学染色观察IR、胰岛素受体底物-1(insulin receptor substrate, IRS-1)、Akt、B淋巴细胞瘤／白血病基因-2(B cell lymphoma/leukemia-2,Bcl-2)和环磷腺苷反应元件结合蛋白(cAMP response element binding protein, CREB)的表达水平。
8.统计学分析。所有数据应用SPSS17.0或SPSS13.0软件进行统计分析,数据分析的结果采用均数土标准差(mean±SD)表示。多组之间的差异采用单因素方差分析(analysis of variance, ANOVA)进行比较,两组之间的差异用Tukey's检验进行比较。以p<0.05表示差异具有统计学意义。
研究结果
1.Aβ1-42寡聚体的鉴定结果：在透射电镜下观察,发现Aβ1-42寡聚体形态比较一致,大小比较均匀,呈规则的球形或椭球形,直径为12-25nm,且未在寡聚体溶液中检测到纤丝状物质形成。
2.星形胶质细胞对Aβ1-42寡聚体的反应：GFAP的mRNA水平和蛋白水平在Aβ1-42寡聚体的作用下均升高(p<0.05),且两者均与Aβ1-42寡聚体的浓度呈剂量依赖性；形态学研究结果显示星形胶质细胞呈反应性增生,表现为胞体和胞核均增大,突起肥大,细胞数量无明显改变。
3.星形胶质细胞insulin/Akt/EAAT通路相关蛋白的表达及活性检测结果：①Aβ1-42寡聚体降低星形胶质细胞IR的mRNA水平(p<0.05),胰岛素对此无影响(p>0.05)；随Aβ1-42寡聚体浓度升高,IR-α、IR-β和p-IR的表达均逐渐减少,而胰岛素能逆转这三种蛋白下降的趋势(p<0.05)。②Akt的mRNA水平和总蛋白水平均不受Aβ1-42寡聚体和胰岛素影响(P＞0.05)；随Aβ1-42寡聚体浓度升高,p-Akt表达减低,而胰岛素则能增加p-Akt的水平(p<0.05)。③在Ap1-42寡聚体和胰岛素的作用下,mTOR的mRNA和总蛋白水平均未改变(P＞0.05)；而p-mTOR的表达随Aβ1-42寡聚体浓度升高而降低(p<0.05),胰岛素能逆转减少的p-mTOR(p<0.05)。④在Aβ1-42寡聚体和胰岛素作用下,EAAT1和EAAT2的mRNA水平保持不变(p>0.05)；而在Aβ1-42寡聚体作用下,EAAT1和EAAT2的总蛋白水平下降(p<0.05)；在胰岛素的刺激下,与相应对照组相比,III组、IV组和V组的EAAT1和EAAT2的蛋白水平均增加(p<0.05)
4.星形胶质细胞活性的测定结果：Aβ1-42寡聚体在浓度为100nmol/L和Iμmol/L时都能导致星形胶质细胞的活性下降(p<0.05)。而与对照组相比,胰岛素的刺激则能增强细胞的活性(p<0.05)
5.动物的行为学评估结果：定向航行实验中大鼠的逃避潜伏期能反应其学习能力,而空间探索实验中大鼠经过平台所在位置的次数、寻找平台的时间比例和路程比例则能反应其记忆能力。①AD组大鼠逃避潜伏期为87.40±6.70s,比NS组(15.23±4.65s,p<0.05)和C组(14.00±6.01s,p<0.05)均明显延长,NS组与C组的逃避潜伏期无明显差异(p>0.05)。②AD组大鼠经过原平台所在位置的次数比NS组和C组明显减少(p<0.05)。③AD组大鼠寻找平台所在位置的时间比例(AD组vs.NS组是0.24±0.09s vs.0.34±0.07s,p<0.05；AD组vs.C组是0.24±0.09s vs.0.35±0.01s,p<0.05)和路程比例(AD组vs.NS组是0.27±0.05vs.0.35±0.03,p<0.05；AD组vs.C组是0.27±0.05vs.0.35±0.06,p<0.05)也均要比NS组和C组显著减少；而在NS组和C组中,大鼠寻找平台所用的时间比例和路程比例也均无明显差异(p>0.05)。
6.大鼠海马神经元胰岛素信号通路相关蛋白的检测结果：①Aβ1-42寡聚体降低海马神经元IR的表达(AD组vs.NS组是0.25±O.02vs.0.38±0.03,p<0.05；AD组vs.C组是0.25±0.02vs.0.40±0.02,p<0.05)。②IRS-1在AD组的表达水平明显比NS组和C组低(AD组vs.NS组是0.22±0.02vs.0.35±0.03,p<0.05；AD组vs.C组是0.22±0.02vs.0.29±0.06,p<0.05),而NS组与C组的差异无统计学意义(p>0.05)。③Akt在AD组的表达水平比NS组(0.20±0.03vs.0.37±0.03,p<0.05)和C组(0.20±0.03vs.0.38±0.03,p<0.05)均减少,在NS组与C组无差异(p>0.05)。④Aβ1-42寡聚体使Bc1-2表达降低(AD组vs.NS组是0.20±0.02vs.0.41±0.04,p<0.05；AD组vs.C组是0.20±0.02vs.0.40±0.06,p<0.05)。⑤AD组大鼠海马神经元CREB的表达与NS组(AD组vs.NS组是0.43±0.03vs.0.40±0.03)和C组(AD组vs.C组是0.43±0.03vs.0.41±0.03)相比无明显差异(p>0.05)。
研究结论
1.采用改良的方法制备Aβ1-42寡聚体能稳定储存,透射电镜鉴定结果符合实验要求,可用于AD模型的制备。
2.Aβ1-42寡聚体对星形胶质细胞和神经元均能造成毒性损害,侧脑室持续微量泵入Aβ1-42寡聚体的大鼠出现学习记忆能力下降。
3.星形胶质细胞上存在insulin/Akt/EAAT通路,且Aβ1-42寡聚体在浓度为100nmol/L时就能使该信号通路相关底物表达异常；AD大鼠海马神经元胰岛素信号转导通路也能在Aβ1-42寡聚体的作用下发生紊乱。即星形胶质细胞和神经元上的胰岛素信号通路紊乱是AD发病的重要机制之一。
4.尽管慢性持续性高血浆胰岛素水平对脑组织有害,但100nmol/L的胰岛素作用于星形胶质细胞30min能保护insulin/Akt/EAAT通路,且对该通路激活有重要意义,即以正确的途径给予合适浓度的胰岛素可能对AD起治疗作用。
Background
Alzheimer's disease (AD), an age-related neurodegenerative disorder, becomes the most common form of dementia among old people. Clinically, AD is characterized by the insidious onset and gradual progression of cognitive decline, psychiatric symptoms and loss in activity of daily living. It is a fatal cause of human death with a high incidence just inferior to the cardiovascular disease and cancer, increasingly becoming an important threat to the health of the elderly. AD is a heterogeneous disease caused by several factors, such as depositing of beta-amyloid protein (Aβ), disturbances of neuronal survival pathways, disorders of glucose metabolism, mitochondrial damage, oxidative stress and inflammation which are involved in the development of the disease. Although the pathogenesis of AD is still elusive, there is an ample consensus that the abnormal Aβ deposition in the central nerves system (CNS) plays the most crucial role, especially Aβ1-42in the form of soluble oligomers, and other pathological changes and pathogenic pathways may be secondary to the result of excessive deposition of Aβ.
Brain insulin signaling pathway involves in energy metabolism and neuroendocrine. It is critical to regulate glucose and lipid metabolism, modulate synaptic genesis and remodeling, promote cell survival and growth, control inflammation, maintain mitochondrial function and improve learning and memory. The destruction of insulin signaling pathway contributes not only to diabetes and obesity but also to neurodegeneration and cognitive decline and it has been demonstrated that its injury can increase the risk of dementia. Furthermore, mammalian target of rapamycin (mTOR), one of the most important downstream targets of protein kinase B (PKB/Akt), is able to regulate the glutamate transporter expression in astrocytes. Through interactions, mTOR and Akt could also regulate synaptic activity and memory formation. Therefore, enhancing the activity of insulin signaling pathway plays an important role in neuroprotection and cognitive improvement.
Astrocytes are the major glial components providing a nurturing environment for neurons in the CNS. To be specific, they contribute to synaptic functions, transfer and store information, participate in cognition, produce trophic factors, remove toxins and debris, maintain redox potential and regulate concentration of neurotransmitter and ion. Noteworthy, astrocytes express abundant insulin receptors (IRs) and might be influenced by insulin. Glutamate, the main excitatory neurotransmitter in the brain, plays a key role in learning and memory. It is released into the synaptic cleft during the neuronal action and rapidly taken up by the excitatory amino acid transporters (EAATs), among which EAAT1and EAAT2preferentially located in astrocytes are responsible for clearing the majority glutamate. Consequently, malfunctions of the EAATs may lead to aberrant glutamate accumulation and neuron injury known as excitotoxicity, which is also the reason for several neurodegenerative disorders including AD.
Objective
The aim of this study is as follows:first, determine the reaction of astrocytes to Aβ1-42oligomers; second, explore whether the insulin/Akt/EAAT signaling is existed in human astrocytes and whether could be influenced by Aβ1-42oligomers and insulin; third, explore the behavioral changes of the AD model rats and explore the effect of Aβ1-42oligomers to the cellular signaling proteins of insulin signaling transduction pathway in the hippocampal neurons. Overall, we aimed to further explore the pathogenesis of AD and its feasible treatment.
Methods
1. Preparation and determination of the Aβ1-42oligomers. We prepared the Aβ oligomers using a basic method and an improved method, respectively. But the Aβ oligomers prepared by the improved method were superior due to its relatively stability in short term. Briefly, human synthetic Aβ1-42lyophilized powder was returned to room temperature firstly and suspended in hexafluoro-isopropanol (HFIP) to make it into monomer followed by further dissolving with dimethyl sulfoxide (DMSO) to produce a homogenous suspension. Subsequently, the Ap-DMSO solution was resuspended with SDS-PBS and incubated at4℃for24h. Ultimately, the solution was diluted with PBS and continually incubated at4℃for2weeks, which was then diluted to the specific concentration with DMEM. The characterization of Aβ1-42oligomers were determined using a transmission electron microscopy (TEM).
2. Groups of astrocytes and drug intervention. Astrocytes were divided into6groups which were treated with drugs after the starvation:control group and groups I to V. In brief, the control group (group C) was the negative control which was treated with DMEM without fetal bovine serum (FBS); group I was treated with100nmol/L Aβ1-42oligomers; group II was treated with1μmol/L Aβ1-42oligomers. All of the three groups were cultured for24h at the atmosphere of5%CO2and37℃. Groups III, IV and V were treated with100nM human recombinant insulin for30min after the treatments with DMEM vehicle,100nM Aβ1-42oligomers and1μM Aβ1-42oligomers, respectively.
3. Observing the reaction of astrocytes to the drugs and detecting expression and activity of the cellular signaling proteins in insulin/Akt/EAAT signaling pathway. First, we determined the mRNA of glial fibrillary acidic protein (GFAP) with real time RT-PCR, determined the GFAP protein with Western blot, and then observed the changes of cell morphology by immunofluorescence staining. Second, we detected the mRNA of IR, Akt, mTOR, EAAT1and EAAT2, detected the protein expression of IR-α, IR-β, phosphorylation of insulin receptor (p-IR, Y1361), Akt, phosphorylation of protein kinase B (p-Akt, S473), mTOR, phosphorylation of mammalian target of rapamycin (p-mTOR, S2448), EAAT1and EAAT2. Third, we further observed the expression of IR-a, IR-(3and p-IR by immunofluorescence staining.
4. Detecting the cell viability of astrocytes. The cell viability of astrocytes treated with Aβ1-42oligomers and insulin was examined through conversing the methyl thiazolyl tetrazolium (MTT) to colored formazan crystals.
5. Groups of animals and preparation of AD rat models.50adult male Wistar rats (3-4months old, healthy and weighing225±25g) were used.5rats were eliminated by behavioral tests preoperatively, and the remaining45rats were divided into three groups, namely the control group (group C, rats only underwent surgery, but without any injection, n=15); the normal saline group (NS group, slow and continuous infusion of normal saline into the lateral ventricle of rats, n=15) and the Aβ1-42oligomers injection group (AD group, slow and continuous injection of Aβ1-42oligomers into the lateral ventricle of rats, n=15). Briefly, the micro-osmotic pump was filled with Aβ1-42oligomer solution or normal saline firstly, and then the various parts were assembled with the micro-osmotic pump, which was implanted into the lateral ventricle with the help of a stereotaxic instrument. Adjust the flow regulator to inject Aβ1-42oligomer solution or normal saline slowly and continuously (3μL/day for30days). All operations followed the aseptic principles. Do neurological assessment until the rats were conscious and80,000units of penicillin were injected to all rats for3days routinely.
6. Test of behavior. The Morris water maze (MWM) test was used to evaluate the learning and memory function of rats at the end of the drug administration. Acquisition trials were done at the31-34th days after the surgery and probe trials were done at the35th day. The platform was placed in the southern quadrant and the escape latency that the time needed to reach the platform in acquisition trials was recorded and analyzed. Then the times passing through the original position of the platform in probe trials was recorded and analyzed; the residence time and the swimming distance in the south quadrant in probe trials was recorded and analyzed; the ratio of the residence time in the south quadrant to the total time was recorded and analyzed; and also the ratio of the swimming distance in the south quadrant to the total swimming distance in probe trials was recorded and analyzed.
7. Detecting the cellular signaling proteins of insulin signaling pathway in hippocampal neurons. Rats were anesthetized and the hippocampus was drawn and fixed. And then, the proteins of IR, insulin receptor substrate-1(IRS-1), Akt, B cel llymphoma/leukemia-2(Bcl-2) and cAMP response element binding protein (CREB) were detected by immunohistochemistry.
8. Statistical analysis. Results are expressed as mean±SD. To determine the significance of difference among various groups, the statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's test using SPSS17.0.p<0.05was considered statistically significant in all tests.
Results
1. Identification of Aβ1-42oligomers. The Aβ1-42oligomers were regular and uniform spheres with a diameter of12-25nm, and no fibrillar aggregates were present in the oligomer solution.
2. Reaction of astrocytes to the Aβ1-42oligomers. The mRNA level of GFAP was elevated under the treatment of Aβ1-42oligomers (p<0.05) and the protein level of GFAP was also increased (p<0.05). Moreover, both mRNA and protein levels of GFAP were changed by Aβ1-42oligomers in a dose-dependent manner. Besides, our morphological study showed that astrocytes were activated by Aβ1-42oligomers, namely enlarged cell bodies and nuclei, hypertrophic processes, and increased GFAP expression. However, there was an absence of cell proliferation.
3. Changes of expression and activity of the cellular signaling proteins of insulin/Akt/EAAT signaling pathway in astrocytes.①The mRNA encoding IR was decreased by Aβ1-42oligomers in a dose-dependent manner (p<0.05), but insulin had no effect on IR mRNA (p>0.05). In addition, the Western blot result showed that lower expressions of IR-a (p<0.05), IR-β (p<0.05) and p-1R at Tyrl361(p<0.05) were potentiated with concentrations of the Aβ1-42oligomers. However, we found a significant effect on IR-a, IR-β and phosphorylation of IR after exposure to insulin compared with their respective control groups(p<0.05).②Both mRNA and protein levels of total Akt were not altered by Aβ1-42oligomers and insulin (p>0.05). However, Akt phosphorylation at Ser473was significantly decreased by the soluble Aβ1-42oligomers (p<0.05). Moreover, we also observed that insulin enhanced the phosphorylation of Akt compared with the control groups (p<0.05).③Aβ1-42oligomers and insulin had no effect on mRNA and protein levels of mTOR (p>0.05). However, the expression of p-mTOR was decreased by the Aβ1-42oligomers (p<0.05) and insulin could reverse the reduction in p-mTOR (p<0.05).④Aβ1-42oligomers and insulin had no effect on the mRNA levels of EAAT1and EAAT2(p>0.05). However, the protein expressions of EAAT1and EAAT2were both lowered by the Aβ1-42oligomers (p<0.05), whereas they were increased by insulin in group III, group IV and group V compared with the control groups (p<0.05).
4. Cell viability of the astrocytes. Incubation with Aβ1-42oligomers at100nmol/L and lμmol/L for24h caused a significant decrease of MTT reduction (p<0.05), whereas insulin exposure induced an increase of MTT reduction compared with the control groups (p<0.05).
5. Assessment of animal behavior. The escape latency in the acquisition trials could react the learning ability of rats and the times of finding the location of the platform, the ratio of time in the south quadrant to the total time and the ratio of distance in the south quadrant to the total distance to find the platform in the probe trials could react the memory capacity of rats.①The escape latency of AD rats was87.40±6.70s, which was significant longer than the NS group (15.23±4.65s, p<0.05) and the control group (14.00±6.01s, p<0.05); however, there was no significant difference between the NS group and the control group (p>0.05).②The frequency of passing through the original position on the platform in AD group were significantly less than the NS group and the control group (p<0.05,p<0.05).③The ratio of time (AD group vs. NS group:0.24±0.09s vs.0.34±0.07s,p<0.05; AD group vs. group C:0.24±0.09s vs.0.35±0.01s,p<0.05) and the ratio of distance (AD group vs. NS group:0.27±0.05vs.0.35±0.03, p<0.05; AD group vs. group C:0.27±0.05vs.0.35±0.06, p<0.05) to find the original position on the platform in the AD group were also significantly less than the NS group and the control group, while no significant difference was detected between the NS group and the control group (p>0.05).
6. Changes of the cellular signaling proteins of insulin signaling pathway in hippocampal neurons:①The expression of IR was reduced by Aβ1-42oligomers (AD group vs. NS group:0.25±0.02vs.0.38±0.03, p<0.05; AD group vs. group C:0.25±0.02vs.0.40±0.02, p<0.05).②The level of IRS-1protein in AD group was significantly lower than the NS group and group C (AD group vs. NS group:0.22±0.02vs.0.35±0.03, p<0.05; AD group vs. group C:0.22±0.02vs.0.29±0.06, p<0.05), while no significant difference was found between the NS group and the group C (p>0.05).③The amount of Akt protein in AD group was significantly lower than the NS group (0.20±0.03vs.0.37±0.03, p<0.05) and the group C (0.20±0.03vs.0.38±0.03,p<0.05), while there is no significant difference between the NS group and the group C (p>0.05).④The protein expression of Bcl-2was decreased by Aβ1-42oligomers (AD group vs. NS group:0.20±0.02vs.0.4±0.04,p<0.05; AD group vs. group C:0.20±0.02vs.0.40±0.06,p<0.05).⑤The level of CREB in AD group was similar with the NS group (AD group vs. NS group:0.43±0.03vs.0.40±0.03) and the group C (AD group vs. group C:0.43±0.03vs.0.41±0.03).
Conclusions
1. The modified method for preparing oligomeric Aβ1-42is stable for storage and viable in the preparation of AD models.
2. Aβ1-42oligomers are toxic to the astrocytes and hippocampal neurons. The continuous injection of Aβ1-42oligomers to the lateral ventricle could decrease the ability of learning and memory in rats.
3. Insulin/Akt/EAAT signaling pathway exists in the astrocytes and Aβ1-42oligomers at100nmol/L can induce abnormal expressions of the cellular signaling proteins in the signaling pathway. The insulin signaling transduction pathway of hippocampal neurons in AD rat can be disturbed by the Aβ1-42oligomers. Overall, disorders of the insulin signaling transduction pathway in astrocytes and neurons may be one of the important pathogenesis of AD.
4. Although chronic and persistent high level of insulin in plasma is harmful to brain tissue, insulin at100nmol/L could protect insulin/Akt/EAAT signaling pathway in astrocytes after30min. In addition, it is also very important for the activity of the signaling pathway. This indicates that a correct way to give appropriate concentration of insulin might give a therapeutic effect on AD.
阿尔茨海默病(Alzheimer's disease, AD)是一种神经退行性疾病,也是老年人痴呆中最常见的类型,表现为进行性加重的认知功能障碍和神经精神症状及生活能力的逐渐丧失。该病发病率高,是继心脑血管疾病和肿瘤之后导致人类死亡的主要原因,正日益成为对老年人健康的重要威胁。AD是一种多因素共同作用而致的复杂异质性疾病,淀粉样蛋白(beta-amyloid protein, Aβ)沉积、神经存活通路紊乱、糖代谢障碍、线粒体损伤、氧化应激和炎症等均参与疾病的发生发展。其中,Ap沉积是公认的“源头”,尤其以Aβ1-42寡聚体毒性最强,而其他的病理改变和致病途径可能是Aβ过量沉积的继发结果。
胰岛素信号通路参与能量代谢和神经内分泌两个过程,能调节糖脂代谢、突触形成及重塑、细胞存活和生长、炎症反应、线粒体功能和学习记忆等。该通路破坏不仅与糖尿病和肥胖等疾病相关,而且与神经退行性变及认知功能下降密不可分,且已经证实该通路损伤后能增加痴呆的发病风险。哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)是蛋白激酶B(protein kinase B, Akt/PKB)的下游底物之一,并且能调节星形胶质细胞谷氨酸转运体的表达。两者还能通过相互作用来调节突触活动及记忆形成。因此,增强胰岛素信号通路的活性能起到神经保护及改善认知的作用。
星形胶质细胞是脑组织的支持细胞,为神经元提供稳定的内环境。具体而言,星形胶质细胞能维持突触的正常功能、转移和储藏信息、参与认知、产生营养因子、移除毒素和代谢产物、维持正常的氧化还原电势以及调节神经递质和离子的浓度等。最重要的是,星形胶质细胞表达有大量胰岛素受体(insulin receptor, IR),并能受胰岛素的影响。谷氨酸是中枢神经系统(central nervous system, CNS)中最重要的兴奋性神经递质,在学习和记忆中起举足轻重的作用。谷氨酸在神经元活动时释放到突触间隙中,然后被星形胶质细胞上的兴奋性氨基酸转运体(excitatory amino acid transporters, EAATs)快速重吸收。该转运体被破坏的直接后果是导致谷氨酸在细胞外过度蓄积而对神经元产生兴奋性毒性损伤,而这也是多种神经退行性疾病包括AD的发病机制之一
研究目的
观察Aβ1-42寡聚体对人星形胶质细胞的影响,探索人星形胶质细胞上是否存在insul in/Akt/EAAT通路,以及Aβ1-42寡聚体和胰岛素对该通路蛋白表达水平和活性的影响作用；同时还观察Aβ1-42寡聚体侧脑室灌注AD模型大鼠的行为学改变及海马神经元胰岛素信号转导通路相关蛋白表达的变化；进一步寻找AD的发病机制及有效的干预靶点。
研究方法
1.Aβ1-42寡聚体的制备和鉴定。我们分别使用基础的方法和改良的方法制备Aβ1-42寡聚体,后者能在短时期内相对稳定保存。具体而言,首先,将Aβ1-42冻干粉与室温平衡后加入六氟异丙醇(hexafluoro-isopropanol, HFIP)使其单体化,再用无水二甲基亚砜(dimethyl sulfoxide, DMSO)溶解Aβ1-42肽膜。然后,使用SDS-PBS重悬Ap-DMSO溶液并于4℃孵育24h。最后,用PBS稀释重悬液,再于4℃孵育2周后用不含血清的DMEM稀释至实验所需的浓度。Ap1-42寡聚体制备完成后在透射电镜下进行观察和鉴定。
2.细胞分组和药物干预。星形胶质细胞共分为6组,待饥饿处理结束后即加药。C组为空白对照组,即给予无胎牛血清(fetal bovine serum, FBS)的DMEM;I组给予100nmol/L的Aβ1-42寡聚体溶液；II组给予1μmol/L的Aβ1-42寡聚体溶液。将三组细胞置于37℃、饱和湿度、5%CO2含量的恒温细胞培养箱中培养24h。后三组是在前三组的基础上均再给予100nmol/L人合成胰岛素作用30min,即III组为给予无FBS的DMEM培养24h后再给予100nmol/L胰岛素作用30min;Ⅳ组为给予100nmol/L的Aβ1-42寡聚体溶液培养24h后再给予100nmol/L胰岛素作用30min;Ⅴ组为给予1μmol/L的Aβ1-42寡聚体溶液培养24h后再给予100nmol/L胰岛素作用30mmin。
3.观察星形胶质细胞对药物的反应及检测insulin/Akt/EAAT通路相关蛋白的表达和活性。首先,用荧光定量RT-PCR和Western blot检测胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)的mRNA和蛋白水平,再通过免疫荧光染色的方法观察细胞形态变化。然后,使用荧光定量RT-PCR检测IR, Akt、 mTOR、EAAT1和EAAT2的mRNA水平；使用Western blot的方法检测IR-α IR-β、磷酸化胰岛素受体(phosphorylation of insulin receptor, p-IR, Y1361)、Akt、磷酸化蛋白激酶B (phosphorylation of protein kinase B, p-Akt, S473) mTOR、磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylation of mammalian target of rapamycin, p-mTOR, S2448), EAAT1和EAAT2的蛋白水平；使用免疫荧光染色的方法进一步观察IR-α、IR-β口p-IR在细胞内的表达情况。
4.星形胶质细胞活性的检测。使用MTT的实验方法检测星形胶质细胞在Aβ1-42寡聚体和胰岛素作用下细胞活性的变化。
5.动物分组和AD动物模型的建立。3-4月龄的健康成年雄性Wistar大鼠50只,体重为225±25g。术前行为学检测淘汰5只,将剩余45只大鼠分为3组。即正常对照组(C组,大鼠仅行手术而未行任何注射)、生理盐水组(NS组,向大鼠侧脑室内缓慢持续泵入生理盐水)和Aβ1-42寡聚体注射组(AD组,向大鼠侧脑室内缓慢持续泵入Aβ1-42寡聚体),各组动物数量均为15只(n=15)。先将Aβ1-42寡聚体溶液或者生理盐水注满微渗泵后再把各部分装置组装起来,并在脑立体定位仪的作用下植入微渗泵。调节流量调节器,使Aβ1-42寡聚体溶液或者生理盐水缓慢持续泵入,每天泵入3μL,连续给药30天。所有操作均遵循无菌操作的原则。术后待大鼠清醒后进行神经功能评定,并常规给予肌肉注射青霉素8万单位以防止切口感染,连续给药3天。
6.行为学检测。给药结束后行Morris水迷宫实验评估大鼠的学习和记忆能力。术后第31-34天进行定向航行实验,第35天进行空间探索实验。将平台置于南象限(随机预设),记录分析每只大鼠的逃避潜伏期,即在定向航行实验时大鼠到达平台的时间；记录分析空间探索实验时大鼠经过平台原位置的次数；记录分析空间探索实验时大鼠在南象限停留的时间；记录分析空间探索实验时大鼠在南象限游动的路程；计算空间探索实验时大鼠在南象限游动的时间及路程占总时间和总路程的比率。
7.大鼠海马神经元胰岛素信号通路相关蛋白表达的检测。麻醉大鼠,取材、固定海马组织后行免疫组织化学染色观察IR、胰岛素受体底物-1(insulin receptor substrate, IRS-1)、Akt、B淋巴细胞瘤／白血病基因-2(B cell lymphoma/leukemia-2,Bcl-2)和环磷腺苷反应元件结合蛋白(cAMP response element binding protein, CREB)的表达水平。
8.统计学分析。所有数据应用SPSS17.0或SPSS13.0软件进行统计分析,数据分析的结果采用均数土标准差(mean±SD)表示。多组之间的差异采用单因素方差分析(analysis of variance, ANOVA)进行比较,两组之间的差异用Tukey's检验进行比较。以p<0.05表示差异具有统计学意义。
研究结果
1.Aβ1-42寡聚体的鉴定结果：在透射电镜下观察,发现Aβ1-42寡聚体形态比较一致,大小比较均匀,呈规则的球形或椭球形,直径为12-25nm,且未在寡聚体溶液中检测到纤丝状物质形成。
2.星形胶质细胞对Aβ1-42寡聚体的反应：GFAP的mRNA水平和蛋白水平在Aβ1-42寡聚体的作用下均升高(p<0.05),且两者均与Aβ1-42寡聚体的浓度呈剂量依赖性；形态学研究结果显示星形胶质细胞呈反应性增生,表现为胞体和胞核均增大,突起肥大,细胞数量无明显改变。
3.星形胶质细胞insulin/Akt/EAAT通路相关蛋白的表达及活性检测结果：①Aβ1-42寡聚体降低星形胶质细胞IR的mRNA水平(p<0.05),胰岛素对此无影响(p>0.05)；随Aβ1-42寡聚体浓度升高,IR-α、IR-β和p-IR的表达均逐渐减少,而胰岛素能逆转这三种蛋白下降的趋势(p<0.05)。②Akt的mRNA水平和总蛋白水平均不受Aβ1-42寡聚体和胰岛素影响(P＞0.05)；随Aβ1-42寡聚体浓度升高,p-Akt表达减低,而胰岛素则能增加p-Akt的水平(p<0.05)。③在Ap1-42寡聚体和胰岛素的作用下,mTOR的mRNA和总蛋白水平均未改变(P＞0.05)；而p-mTOR的表达随Aβ1-42寡聚体浓度升高而降低(p<0.05),胰岛素能逆转减少的p-mTOR(p<0.05)。④在Aβ1-42寡聚体和胰岛素作用下,EAAT1和EAAT2的mRNA水平保持不变(p>0.05)；而在Aβ1-42寡聚体作用下,EAAT1和EAAT2的总蛋白水平下降(p<0.05)；在胰岛素的刺激下,与相应对照组相比,III组、IV组和V组的EAAT1和EAAT2的蛋白水平均增加(p<0.05)
4.星形胶质细胞活性的测定结果：Aβ1-42寡聚体在浓度为100nmol/L和Iμmol/L时都能导致星形胶质细胞的活性下降(p<0.05)。而与对照组相比,胰岛素的刺激则能增强细胞的活性(p<0.05)
5.动物的行为学评估结果：定向航行实验中大鼠的逃避潜伏期能反应其学习能力,而空间探索实验中大鼠经过平台所在位置的次数、寻找平台的时间比例和路程比例则能反应其记忆能力。①AD组大鼠逃避潜伏期为87.40±6.70s,比NS组(15.23±4.65s,p<0.05)和C组(14.00±6.01s,p<0.05)均明显延长,NS组与C组的逃避潜伏期无明显差异(p>0.05)。②AD组大鼠经过原平台所在位置的次数比NS组和C组明显减少(p<0.05)。③AD组大鼠寻找平台所在位置的时间比例(AD组vs.NS组是0.24±0.09s vs.0.34±0.07s,p<0.05；AD组vs.C组是0.24±0.09s vs.0.35±0.01s,p<0.05)和路程比例(AD组vs.NS组是0.27±0.05vs.0.35±0.03,p<0.05；AD组vs.C组是0.27±0.05vs.0.35±0.06,p<0.05)也均要比NS组和C组显著减少；而在NS组和C组中,大鼠寻找平台所用的时间比例和路程比例也均无明显差异(p>0.05)。
6.大鼠海马神经元胰岛素信号通路相关蛋白的检测结果：①Aβ1-42寡聚体降低海马神经元IR的表达(AD组vs.NS组是0.25±O.02vs.0.38±0.03,p<0.05；AD组vs.C组是0.25±0.02vs.0.40±0.02,p<0.05)。②IRS-1在AD组的表达水平明显比NS组和C组低(AD组vs.NS组是0.22±0.02vs.0.35±0.03,p<0.05；AD组vs.C组是0.22±0.02vs.0.29±0.06,p<0.05),而NS组与C组的差异无统计学意义(p>0.05)。③Akt在AD组的表达水平比NS组(0.20±0.03vs.0.37±0.03,p<0.05)和C组(0.20±0.03vs.0.38±0.03,p<0.05)均减少,在NS组与C组无差异(p>0.05)。④Aβ1-42寡聚体使Bc1-2表达降低(AD组vs.NS组是0.20±0.02vs.0.41±0.04,p<0.05；AD组vs.C组是0.20±0.02vs.0.40±0.06,p<0.05)。⑤AD组大鼠海马神经元CREB的表达与NS组(AD组vs.NS组是0.43±0.03vs.0.40±0.03)和C组(AD组vs.C组是0.43±0.03vs.0.41±0.03)相比无明显差异(p>0.05)。
研究结论
1.采用改良的方法制备Aβ1-42寡聚体能稳定储存,透射电镜鉴定结果符合实验要求,可用于AD模型的制备。
2.Aβ1-42寡聚体对星形胶质细胞和神经元均能造成毒性损害,侧脑室持续微量泵入Aβ1-42寡聚体的大鼠出现学习记忆能力下降。
3.星形胶质细胞上存在insulin/Akt/EAAT通路,且Aβ1-42寡聚体在浓度为100nmol/L时就能使该信号通路相关底物表达异常；AD大鼠海马神经元胰岛素信号转导通路也能在Aβ1-42寡聚体的作用下发生紊乱。即星形胶质细胞和神经元上的胰岛素信号通路紊乱是AD发病的重要机制之一。
4.尽管慢性持续性高血浆胰岛素水平对脑组织有害,但100nmol/L的胰岛素作用于星形胶质细胞30min能保护insulin/Akt/EAAT通路,且对该通路激活有重要意义,即以正确的途径给予合适浓度的胰岛素可能对AD起治疗作用。
Background
Alzheimer's disease (AD), an age-related neurodegenerative disorder, becomes the most common form of dementia among old people. Clinically, AD is characterized by the insidious onset and gradual progression of cognitive decline, psychiatric symptoms and loss in activity of daily living. It is a fatal cause of human death with a high incidence just inferior to the cardiovascular disease and cancer, increasingly becoming an important threat to the health of the elderly. AD is a heterogeneous disease caused by several factors, such as depositing of beta-amyloid protein (Aβ), disturbances of neuronal survival pathways, disorders of glucose metabolism, mitochondrial damage, oxidative stress and inflammation which are involved in the development of the disease. Although the pathogenesis of AD is still elusive, there is an ample consensus that the abnormal Aβ deposition in the central nerves system (CNS) plays the most crucial role, especially Aβ1-42in the form of soluble oligomers, and other pathological changes and pathogenic pathways may be secondary to the result of excessive deposition of Aβ.
Brain insulin signaling pathway involves in energy metabolism and neuroendocrine. It is critical to regulate glucose and lipid metabolism, modulate synaptic genesis and remodeling, promote cell survival and growth, control inflammation, maintain mitochondrial function and improve learning and memory. The destruction of insulin signaling pathway contributes not only to diabetes and obesity but also to neurodegeneration and cognitive decline and it has been demonstrated that its injury can increase the risk of dementia. Furthermore, mammalian target of rapamycin (mTOR), one of the most important downstream targets of protein kinase B (PKB/Akt), is able to regulate the glutamate transporter expression in astrocytes. Through interactions, mTOR and Akt could also regulate synaptic activity and memory formation. Therefore, enhancing the activity of insulin signaling pathway plays an important role in neuroprotection and cognitive improvement.
Astrocytes are the major glial components providing a nurturing environment for neurons in the CNS. To be specific, they contribute to synaptic functions, transfer and store information, participate in cognition, produce trophic factors, remove toxins and debris, maintain redox potential and regulate concentration of neurotransmitter and ion. Noteworthy, astrocytes express abundant insulin receptors (IRs) and might be influenced by insulin. Glutamate, the main excitatory neurotransmitter in the brain, plays a key role in learning and memory. It is released into the synaptic cleft during the neuronal action and rapidly taken up by the excitatory amino acid transporters (EAATs), among which EAAT1and EAAT2preferentially located in astrocytes are responsible for clearing the majority glutamate. Consequently, malfunctions of the EAATs may lead to aberrant glutamate accumulation and neuron injury known as excitotoxicity, which is also the reason for several neurodegenerative disorders including AD.
Objective
The aim of this study is as follows:first, determine the reaction of astrocytes to Aβ1-42oligomers; second, explore whether the insulin/Akt/EAAT signaling is existed in human astrocytes and whether could be influenced by Aβ1-42oligomers and insulin; third, explore the behavioral changes of the AD model rats and explore the effect of Aβ1-42oligomers to the cellular signaling proteins of insulin signaling transduction pathway in the hippocampal neurons. Overall, we aimed to further explore the pathogenesis of AD and its feasible treatment.
Methods
1. Preparation and determination of the Aβ1-42oligomers. We prepared the Aβ oligomers using a basic method and an improved method, respectively. But the Aβ oligomers prepared by the improved method were superior due to its relatively stability in short term. Briefly, human synthetic Aβ1-42lyophilized powder was returned to room temperature firstly and suspended in hexafluoro-isopropanol (HFIP) to make it into monomer followed by further dissolving with dimethyl sulfoxide (DMSO) to produce a homogenous suspension. Subsequently, the Ap-DMSO solution was resuspended with SDS-PBS and incubated at4℃for24h. Ultimately, the solution was diluted with PBS and continually incubated at4℃for2weeks, which was then diluted to the specific concentration with DMEM. The characterization of Aβ1-42oligomers were determined using a transmission electron microscopy (TEM).
2. Groups of astrocytes and drug intervention. Astrocytes were divided into6groups which were treated with drugs after the starvation:control group and groups I to V. In brief, the control group (group C) was the negative control which was treated with DMEM without fetal bovine serum (FBS); group I was treated with100nmol/L Aβ1-42oligomers; group II was treated with1μmol/L Aβ1-42oligomers. All of the three groups were cultured for24h at the atmosphere of5%CO2and37℃. Groups III, IV and V were treated with100nM human recombinant insulin for30min after the treatments with DMEM vehicle,100nM Aβ1-42oligomers and1μM Aβ1-42oligomers, respectively.
3. Observing the reaction of astrocytes to the drugs and detecting expression and activity of the cellular signaling proteins in insulin/Akt/EAAT signaling pathway. First, we determined the mRNA of glial fibrillary acidic protein (GFAP) with real time RT-PCR, determined the GFAP protein with Western blot, and then observed the changes of cell morphology by immunofluorescence staining. Second, we detected the mRNA of IR, Akt, mTOR, EAAT1and EAAT2, detected the protein expression of IR-α, IR-β, phosphorylation of insulin receptor (p-IR, Y1361), Akt, phosphorylation of protein kinase B (p-Akt, S473), mTOR, phosphorylation of mammalian target of rapamycin (p-mTOR, S2448), EAAT1and EAAT2. Third, we further observed the expression of IR-a, IR-(3and p-IR by immunofluorescence staining.
4. Detecting the cell viability of astrocytes. The cell viability of astrocytes treated with Aβ1-42oligomers and insulin was examined through conversing the methyl thiazolyl tetrazolium (MTT) to colored formazan crystals.
5. Groups of animals and preparation of AD rat models.50adult male Wistar rats (3-4months old, healthy and weighing225±25g) were used.5rats were eliminated by behavioral tests preoperatively, and the remaining45rats were divided into three groups, namely the control group (group C, rats only underwent surgery, but without any injection, n=15); the normal saline group (NS group, slow and continuous infusion of normal saline into the lateral ventricle of rats, n=15) and the Aβ1-42oligomers injection group (AD group, slow and continuous injection of Aβ1-42oligomers into the lateral ventricle of rats, n=15). Briefly, the micro-osmotic pump was filled with Aβ1-42oligomer solution or normal saline firstly, and then the various parts were assembled with the micro-osmotic pump, which was implanted into the lateral ventricle with the help of a stereotaxic instrument. Adjust the flow regulator to inject Aβ1-42oligomer solution or normal saline slowly and continuously (3μL/day for30days). All operations followed the aseptic principles. Do neurological assessment until the rats were conscious and80,000units of penicillin were injected to all rats for3days routinely.
6. Test of behavior. The Morris water maze (MWM) test was used to evaluate the learning and memory function of rats at the end of the drug administration. Acquisition trials were done at the31-34th days after the surgery and probe trials were done at the35th day. The platform was placed in the southern quadrant and the escape latency that the time needed to reach the platform in acquisition trials was recorded and analyzed. Then the times passing through the original position of the platform in probe trials was recorded and analyzed; the residence time and the swimming distance in the south quadrant in probe trials was recorded and analyzed; the ratio of the residence time in the south quadrant to the total time was recorded and analyzed; and also the ratio of the swimming distance in the south quadrant to the total swimming distance in probe trials was recorded and analyzed.
7. Detecting the cellular signaling proteins of insulin signaling pathway in hippocampal neurons. Rats were anesthetized and the hippocampus was drawn and fixed. And then, the proteins of IR, insulin receptor substrate-1(IRS-1), Akt, B cel llymphoma/leukemia-2(Bcl-2) and cAMP response element binding protein (CREB) were detected by immunohistochemistry.
8. Statistical analysis. Results are expressed as mean±SD. To determine the significance of difference among various groups, the statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's test using SPSS17.0.p<0.05was considered statistically significant in all tests.
Results
1. Identification of Aβ1-42oligomers. The Aβ1-42oligomers were regular and uniform spheres with a diameter of12-25nm, and no fibrillar aggregates were present in the oligomer solution.
2. Reaction of astrocytes to the Aβ1-42oligomers. The mRNA level of GFAP was elevated under the treatment of Aβ1-42oligomers (p<0.05) and the protein level of GFAP was also increased (p<0.05). Moreover, both mRNA and protein levels of GFAP were changed by Aβ1-42oligomers in a dose-dependent manner. Besides, our morphological study showed that astrocytes were activated by Aβ1-42oligomers, namely enlarged cell bodies and nuclei, hypertrophic processes, and increased GFAP expression. However, there was an absence of cell proliferation.
3. Changes of expression and activity of the cellular signaling proteins of insulin/Akt/EAAT signaling pathway in astrocytes.①The mRNA encoding IR was decreased by Aβ1-42oligomers in a dose-dependent manner (p<0.05), but insulin had no effect on IR mRNA (p>0.05). In addition, the Western blot result showed that lower expressions of IR-a (p<0.05), IR-β (p<0.05) and p-1R at Tyrl361(p<0.05) were potentiated with concentrations of the Aβ1-42oligomers. However, we found a significant effect on IR-a, IR-β and phosphorylation of IR after exposure to insulin compared with their respective control groups(p<0.05).②Both mRNA and protein levels of total Akt were not altered by Aβ1-42oligomers and insulin (p>0.05). However, Akt phosphorylation at Ser473was significantly decreased by the soluble Aβ1-42oligomers (p<0.05). Moreover, we also observed that insulin enhanced the phosphorylation of Akt compared with the control groups (p<0.05).③Aβ1-42oligomers and insulin had no effect on mRNA and protein levels of mTOR (p>0.05). However, the expression of p-mTOR was decreased by the Aβ1-42oligomers (p<0.05) and insulin could reverse the reduction in p-mTOR (p<0.05).④Aβ1-42oligomers and insulin had no effect on the mRNA levels of EAAT1and EAAT2(p>0.05). However, the protein expressions of EAAT1and EAAT2were both lowered by the Aβ1-42oligomers (p<0.05), whereas they were increased by insulin in group III, group IV and group V compared with the control groups (p<0.05).
4. Cell viability of the astrocytes. Incubation with Aβ1-42oligomers at100nmol/L and lμmol/L for24h caused a significant decrease of MTT reduction (p<0.05), whereas insulin exposure induced an increase of MTT reduction compared with the control groups (p<0.05).
5. Assessment of animal behavior. The escape latency in the acquisition trials could react the learning ability of rats and the times of finding the location of the platform, the ratio of time in the south quadrant to the total time and the ratio of distance in the south quadrant to the total distance to find the platform in the probe trials could react the memory capacity of rats.①The escape latency of AD rats was87.40±6.70s, which was significant longer than the NS group (15.23±4.65s, p<0.05) and the control group (14.00±6.01s, p<0.05); however, there was no significant difference between the NS group and the control group (p>0.05).②The frequency of passing through the original position on the platform in AD group were significantly less than the NS group and the control group (p<0.05,p<0.05).③The ratio of time (AD group vs. NS group:0.24±0.09s vs.0.34±0.07s,p<0.05; AD group vs. group C:0.24±0.09s vs.0.35±0.01s,p<0.05) and the ratio of distance (AD group vs. NS group:0.27±0.05vs.0.35±0.03, p<0.05; AD group vs. group C:0.27±0.05vs.0.35±0.06, p<0.05) to find the original position on the platform in the AD group were also significantly less than the NS group and the control group, while no significant difference was detected between the NS group and the control group (p>0.05).
6. Changes of the cellular signaling proteins of insulin signaling pathway in hippocampal neurons:①The expression of IR was reduced by Aβ1-42oligomers (AD group vs. NS group:0.25±0.02vs.0.38±0.03, p<0.05; AD group vs. group C:0.25±0.02vs.0.40±0.02, p<0.05).②The level of IRS-1protein in AD group was significantly lower than the NS group and group C (AD group vs. NS group:0.22±0.02vs.0.35±0.03, p<0.05; AD group vs. group C:0.22±0.02vs.0.29±0.06, p<0.05), while no significant difference was found between the NS group and the group C (p>0.05).③The amount of Akt protein in AD group was significantly lower than the NS group (0.20±0.03vs.0.37±0.03, p<0.05) and the group C (0.20±0.03vs.0.38±0.03,p<0.05), while there is no significant difference between the NS group and the group C (p>0.05).④The protein expression of Bcl-2was decreased by Aβ1-42oligomers (AD group vs. NS group:0.20±0.02vs.0.4±0.04,p<0.05; AD group vs. group C:0.20±0.02vs.0.40±0.06,p<0.05).⑤The level of CREB in AD group was similar with the NS group (AD group vs. NS group:0.43±0.03vs.0.40±0.03) and the group C (AD group vs. group C:0.43±0.03vs.0.41±0.03).
Conclusions
1. The modified method for preparing oligomeric Aβ1-42is stable for storage and viable in the preparation of AD models.
2. Aβ1-42oligomers are toxic to the astrocytes and hippocampal neurons. The continuous injection of Aβ1-42oligomers to the lateral ventricle could decrease the ability of learning and memory in rats.
3. Insulin/Akt/EAAT signaling pathway exists in the astrocytes and Aβ1-42oligomers at100nmol/L can induce abnormal expressions of the cellular signaling proteins in the signaling pathway. The insulin signaling transduction pathway of hippocampal neurons in AD rat can be disturbed by the Aβ1-42oligomers. Overall, disorders of the insulin signaling transduction pathway in astrocytes and neurons may be one of the important pathogenesis of AD.
4. Although chronic and persistent high level of insulin in plasma is harmful to brain tissue, insulin at100nmol/L could protect insulin/Akt/EAAT signaling pathway in astrocytes after30min. In addition, it is also very important for the activity of the signaling pathway. This indicates that a correct way to give appropriate concentration of insulin might give a therapeutic effect on AD.
引文
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