【作者】 林智颖；

【导师】 陈晓春；

【作者基本信息】 福建医科大学 ， 神经病学， 2009， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease,AD)是一种以进行性认知功能损害为主要临床症状的慢性、进行性的神经系统变性疾病。β淀粉样蛋白(β-amyloid protein,Aβ)沉积引起的神经炎性斑块(Neurite plaques,NP)是AD的主要病理特征之一。Aβ具有通过β-折叠的片层结构形成凝聚态的特性,短肽Aβ25-35是其毒性片断。Aβ可通过多种途径发挥神经毒性作用,其中Aβ诱导的胞内[Ca2+]i浓度的升高具有关键性的作用。人参皂苷Rb1为人参的主要活性成分之一,我们前期的实验证实Rb1对AD具有神经保护作用,近期的研究发现人参皂苷Rb1可减少凝聚态Aβ25-35诱导的神经细胞钙浓度从而降低钙蛋白酶(calpain)活性,减轻tau蛋白磷酸化,保持微管稳定性。但是,Rb1减轻细胞内Ca2+浓度的机制仍不清楚,由于电压门控的钙通道及内质网钙库参与的钙调控在Aβ25-35诱导的细胞内钙超载中起重要作用,本研究拟从上述这两方面来探讨Rb1对钙超载影响的可能机制。方法:应用全细胞膜片钳技术通过设定的不同刺激方案记录原代培养7d海马神经元上的电压门控的钙通道电流(voltage-gated calcium channel current ,VGCC)。利用细胞外灌流、细胞内液给药及预孵育等给药方式,同时分别应用各型钙通道特异性阻断剂或各种蛋白激酶的抑制剂,通过比较加药前后或不同组间电流幅度的变化探讨Aβ25-35对VGCC的调控及Rb1对Aβ25-35诱导的钙内流的作用机制。预先用钙高度特异性荧光探针Flou-3/AM负载海马神经元,进行荧光染色,利用激光扫描共聚焦显微镜观察在不同药物干预条件下海马神经元内游离钙离子荧光强度的变化探讨Aβ25-35对内质网钙库调控及Rb1对Aβ25-35诱导的内质网钙库释放的影响。结果:(1)在原代培养的海马神经元上存在多种的钙电流成分(L、N、P/Q、T等),它们具有各自不同的药理学及动力学特性;急性给予10μmolL-1凝聚态Aβ25-35对VGCC没有影响;10μmolL-1凝聚态Aβ25-35预孵育3、6、12、24h后,各组电流均较对照组有不同程度的增加,并以预孵育3h记录到的高电压激活的钙电流(HVA-Ica)变化为著,而预孵育对细胞的膜电容没有影响。(2)L-型钙通道的特异性抑制剂nifedipine完全抑制了凝聚态Aβ25-35诱导的Ca2+通道电流的增加;N型钙通道特异性抑制剂ω-conotoxin GVIA及P/Q型通道特异性的抑制剂ω-agatoxin IVA均无法完全抑制凝聚态Aβ25-35诱导的Ca2+通道电流的增加。PKA抑制剂H-89部分抑制了Aβ预孵育组的电流,而MAPK抑制剂PD98059无法抑制Aβ诱导的电流的增大。(3)2、10、20μM各浓度组凝聚态Aβ25-35均增加了海马神经元胞内[Ca2+]i;IP3R的特异性抑制剂2-APB明显地抑制了胞内[Ca2+]i的增加,兰尼硷受体(RyR)的特异性抑制剂dantrolene却无法抑制。Aβ25-35作用1h后内质网钙容量即有明显下降,在作用24h后降低更为明显。在无细胞外钙情况下,磷脂酶C的抑制剂U73122部分抑制了Aβ25-35诱导的胞内[Ca2+]i的增加。(4)人参皂苷Rb1可以浓度依赖性的方式抑制海马神经元上VGCC,并且抑制了Aβ25-35诱导的VGCC。Nifedipine完全阻断了之前1μMRb1对Aβ25-35诱导的VGCC的抑制,而ω-conotoxin-GVIA、ω-agatoxin IVA等作用前后1μMRb1对Aβ25-35诱导的VGCC的抑制作用没有受到影响;Rb1使Aβ25-35作用的稳态失活曲线向左即超极化方向偏移,而对Aβ25-35诱导的VGCC的激活特性没有影响;cAMP的类似物Forskolin并没有消除10μM Rb1对VGCC的抑制,Rb1对Aβ25-35诱导的VGCC的抑制率亦没有被PKA/PKC抑制剂staurosporine所影响。在无细胞外钙情况下,各浓度组Rb1不能抑制Aβ25-35诱导的细胞内钙荧光强度的增强。结论:凝聚态Aβ25-35可通过PKA系统对细胞膜上特定的钙通道进行磷酸化调控增大VGCC。Aβ25-35可通过IP3途径产生IP3作用于IP3R而引起内质网钙的释放,磷脂酶C的活化可能参与了上述过程。人参皂苷Rb1是一种钙通道阻滞剂,它通过选择性地作用于L-型电压门控的钙通道,影响钙通道的失活过程,即通过加速通道向失活态的转化抑制通道的活性从而抑制了Aβ25-35诱导的VGCC。Rb1对Aβ25-35诱导的细胞内钙的释放没有影响。以上可能是Rb1减轻Aβ25-35诱导的海马神经元钙超载的分子机制,也是人参在抗衰老及在治疗阿尔茨海默病中发挥药理作用的细胞和分子基础。更多还原

【Abstract】 Alzheimer’s disease(AD) is a progressive neurodegenerative disease that is characterized by its typical clinical sympotoms of progressive cognitive impairment. The deposition of amyloidβprotein (Aβ), as insoluble fibrillar aggregates in neuritic plaque, is the dominant histological hallmark of Alzheimer’s disease. Aβtends to form its aggregate state throughβ-sheet lamellar structure, and Aβ25-35 is its toxic fragment. Aβexerts its neurotoxicity through a variety of ways, elevation of intracellular calcium may play a key role of all.Ginsenoside Rb1, one protopanaxadiol type saponin, is one of the most important active compounds of ginseng. Our previous experiments confirmed that Rb1 has neuroprotective effects for AD . Most recently we found that Rb1 could decrease the elevation of intracellular calcium induced by aggregated Aβ25-35 so as to inhibit the activity of calpain and lower the level of phosphorylation of tau and help stabilize microtube.However, the underlie mechanism by which Rb1 decreases the concentration of Ca2+ in cell remains unknown.Because voltage-gated calcium channel and endoplasmic reticulum calcium stores plays an important role in calcium overload in cells induced by Aβ25-35 therefore in this paper we probed into the possible mechanism of Rb1’s effect on calcium overload based on mechanisms of the two sides .Method: VGCC was recorded in primary cultured hippocampal neurons (7 days in vitro, DIV7)by using whole cell configuration of patch clamp technique.Drug was applied by extracellular bath or adding in the pipette solution or pretreatment,and its effect was determined by comparing the amplitude of IHVA before and after the drug application(or among different groups).A variety of calcium channel blockers or specific protein kinase inhibitors were involved in it to examine the effect of Aβ25-35 on VGCC and the possible mechanism of Rb1’s effect.The hippocampal neurons were loaded with calcium-sensitive fluorescent indicator Fluo-3/AM. Intracellular calcium concentration ([Ca2+]i)changes were measured in different conditions of drug intervention by using laser scanning confocal microscopy so as to examine the effect of Aβ25-35 on endoplasmic reticulum calcium stores and the possible mechanism of Rb1’s effect on calcium release of endoplasmic reticulum induced by Aβ25-35.Result: (1)There were multiple types of Ca2+ channel in primary cultured hippocampal neurons (L, N, P / Q, T, etc.), they could be distinguished on the basis of pharmacological and kinetic properties. Acute extracellular exposure of 10μmolL-1 aggregated Aβ25-35 has no effect on VGCC; However ICa of each group(aggregated Aβ25-35 preincubated for 3, 6, 12, 24h) all had augment compared to the control group with a pronounced effect in the preincubated-3h group. Preincubation with aggregated Aβdid not have an effect on whole-cell capacitance of hippocampal neurons compared to the control group.(2)Block of L-type Ca2+ channels by its specific inhibitor nifedipine completely prevent the current augmentation induced by preincubation with aggregated Aβ,however, block of N-type Ca2+ channels or P/Q-type Ca2+ channels by their specific inhibitorsω-conotoxin GVIA andω-agatoxin IVA,respectively,could not prevent the current augmentation induced by preincubation with aggregated Aβ. PKA specific inhibitor,H-89, partially inhibited calcium current in the preincubated group but MAPK specific inhibitor, PD98059, could not .(3) Groups of 2、10、20μM Aβ25-35 all increase [Ca2+]i in hippocampal neurons. 2-APB, an inhibitor of ER Ca2+ release through channels associated to IP3R, was shown to prevent the aggregated Aβ25-35-induced rise of [Ca2+]i, suggesting the involvement of Ca2+ release by ER. Treatment with aggregated Aβ25-35 for 1 h induce a significant decrease in the ER Ca2+ content (P<0.01), which was more pronounced 24 h after the addition of aggregated Aβ25-35. The increase in [Ca2+]i observed in aggregated Aβ25-35-treated cells was prevented by the PLC inhibitor U-73122 in the absence of extracellular Ca2+.(4)Ginsenoside Rb1 inhibited calcium channel current in hippocampal neurons in a dose-dependent manner and also had the inhibitory effect on VGCC induced by Aβ25-35. Nifedipine blocks Ginsenoside Rb1-sensitive VGCC induced by Aβ25-35 completely.However eitherω-conotoxin-GVIA(a selective blocker of N-type channels)orω-agatoxin IVA(a selective blocker of P/Q -type channels)could not dimininish Rb1-sensitive VGCC induced by Aβ25-35. Ginsenoside Rb1 induced a leftward shift of the steady-state inactivation curves of ICa to negative potential in the preincubated group without affecting its activation kinetics.The co-application of 10μmol/L Rb1 and forskolin did not cancel the reduction and the action of 10μmol/L Rb1 was not affected by staurosporine(a PKA / PKC inhibitor).Conclusion:Aggregated Aβ25-35 increased VGCC by regulating the phosphorylation of specific cell membrane calcium channel via PKA. IP3 pathway involved in the early release of Ca2+ from ER induced by Aβ25-35, the activation of PLC may involve in the process. Ginsenoside Rb1 high selectively targeted L-type calcium channel and inhibited channel activity by accelerating it access to the state of inactivation.Rb1 did not affect release of intracellular calcium induced by Aβ25-35. The above may be its molecular mechanisms in alleviating calcium overload induced by Aβ25-35 and the cell basis of ginseng to play pharmacological role in the Anti-Aging and the treatment of Alzheimer’s disease.更多还原