基于Ca2+-CaMKⅣ-CREB信号通路研究染料木素干预阿尔茨海默病机制

Alzheimer's disease (AD) has become one of the main causes of death of old people. Recently, the pathogenesis of AD is closely related to the disturbance of Ca2+-CaMKIV-CREB signal pathway. Traditional Chinese medical science considers the pathogenesis of AD as marrow depletion, brain deficit and mind malajustment and makes the kidney-jing deficiency as a principal part and nourishing kidney to produce marrow as an important way. Soybean can nourish kidney and benefit brain and nourish the bone marrow and genistein is the main active component of isoflavones contained in soybean. We previously found that soybean isoflavones and genistein could make anti-AD effects through improving study and memory ability and reducing the loss of hippocampal neurons.With in vivo and in vitro methods and modern molecular biology experimental techniques such as laser confocal technology, Western blot, Real-time PCR and Immunohistochemistry, the subject, based on Ca2+-CaMKIV-CREB signal pathway, investigates the effects of genistein on the contents or expressions of the key material of Ca2+-CaMKIV-CREB signal pathway in AD models, probes the mechanism of genistein intervening AD and improving study and memory ability and seeks a new method for clinical prevention and treatment of AD.

阿尔茨海默病(AD)已成为老年人主要死因之一。目前认为AD的发病与钙离子（Ca2+）-钙调蛋白依赖性蛋白激酶Ⅳ（CaMKⅣ）-环腺苷酸应答元件结合蛋白（CREB）信号通路失调密切相关。中医认为AD基本病机为髓减脑消、神机失调，以肾精亏虚为本，补肾精生脑髓为其治疗大法。大豆具有补肾益智作用，功能填精益髓，染料木素是大豆中所含异黄酮的主要活性成分。我们既往研究发现，大豆异黄酮及染料木素具有改善学习记忆功能、减少海马神经元丢失等从而发挥防治AD的作用。鉴此，本课题基于Ca2+-CaMKⅣ-CREB信号通路，拟采用体内外研究方法，运用激光共聚焦法、Western blot法、实时荧光定量PCR法、免疫组化等现代分子生物学实验技术手段，观察染料木素对AD模型Ca2+-CaMKⅣ-CREB信号通路关键物质的含量或表达的影响，探讨染料木素防治AD、改善学习记忆功能的作用机制，寻求临床防治AD的新方法。

本课题运用预防手段，采用体内外实验方法，研究染料木素对阿尔茨海默病（AD）的神经保护作用机制与Ca2+-CaMKIV-CREB信号通路的相关性。体内实验时将大鼠随机分为假手术组、模型组、染料木素低中高剂量组、雌激素阳性对照组，预先给药7天后，以D-半乳糖腹腔注射联合Aβ25～35双侧海马注射诱导制备AD大鼠模型，继续给药42天，采用Morris水迷宫实验和跳台实验检测大鼠行为学改变，HE染色观测大鼠海马形态学改变，Tunel法检测细胞凋亡情况。体外实验进行新生鼠海马神经元原代培养，采用MTT法检测细胞活力，流式细胞术检测神经细胞Ca2+浓度。免疫组织化学法和Western blot法检测大鼠海马和原代培养的海马神经元Ca2+-CaMKIV-CREB信号通路相关蛋白的表达。结果发现：（1）Morris水迷宫实验显示染料木素可明显提高AD大鼠的学习记忆能力；（2）HE染色显示染料木素可减少AD大鼠海马神经元的丢失；（3）Tunel染色显示染料木素可减少AD大鼠海马神经元的凋亡；（4）MTT法显示染料木素可提高AD细胞模型的细胞存活率；（5）流式细胞术显示染料木素可减少AD细胞模型Ca2+浓度，减少钙超载；（6）免疫组化和Western blot法显示：染料木素可减少AD动物模型和细胞模型中CaM、CaMKK、p-CaMKⅣ、p-CREB、p-tau，而tau蛋白含量未见明显变化。结论：（1）染料木素可显著提高AD大鼠学习记忆能力；（2）染料木素对AD具有神经保护作用；（3）染料木素的神经保护可能通过减少海马神经元钙超载、下调Ca2+-CaMKIV-CREB信号通路的关键信号物质的表达起作用。本课题的顺利完成，揭示了染料木素预防阿尔茨海默病、改善学习记忆能力的部分作用机制，为临床防治AD提供新的思路和方法。