天麻素早期干预糖尿病大鼠认知功能障碍的实验研究

Cognitive impairment in diabetes （CID）is a serious threat to public health, and therefore it has been a focus of research topic in recent years. The occurrence of CID is difficult to predict as the development process is gradual. Moreover, the molecular and cellular mechanisms leading to CID have remained uncertain. Indeed, there is an apparent lack of information on early diagnosis and effective therapeutic treatment of CID and, hence，when a definite diagnosis is confirmed clinically patients have suffered unduly serious consequences. In view of this, it is desirable to explore for early diagnosis index and search for an effective and early treatment method. It is known that the cognitive function of diabetic rats changes with different degrees of disease severity indicating the involvement of hippocampus. This study is aimed to evaluate the changes of behavioral cognition in diabetic rats using Morris maze. Along with this, gastrodin will be administered to rats with experimentally induced diabetes as a potential early drug intervention. We seek to determine some relevant biomarkers including neurotransmitters and neural signal transduction pathways that have been reported to affect the cognitive function in the hippocampus. To this end, the hippocampus tissue will be examined by electron microscopy, H-E staining, immunofluorescence by confocal laser microscopy, image analysis, western-blot, Real time-PCR and ELISA. In this connection, we will assess the alterations of synaptophysin, ChAT, nNOS, PKA, CaM Ⅱand NF-κB, and to determine the possible molecular mechanisms and correlation between cognitive impairment and gastrodin intervention. The study would help to better understand the development process and mechanism of CID. More importantly, the results would provide an important theoretical basis for exploring early diagnosis index and an effective treatment targeting specifically at the disease.

糖尿病性认知障碍(cognitive impairment in diabetes, CID)严重威胁人民健康，是近年的研究热点。CID发病隐匿、进展缓慢，其早期诊断及治疗仍缺乏有效的手段和方法，确诊时，已出现严重后果。目前急需发掘CID早诊断的有效检测指标，亟待开发CID早治疗的有效方法。本项目拟随病程进展，从早期即开始使用天麻素干预CID病变，在运用Morris水迷宫观测糖尿病大鼠的认知功能变化的基础上，运用电镜、H-E染色、免疫荧光、Real time-PCR、Western blot、ELISA等方法，观察不同病程糖尿病大鼠海马组织病变，深入研究与认知功能密切相关的Synaptophysin、ChAT、nNOS、PKA、CaMⅡ、NF-κB等因子的变化及相互关系，进一步阐明CID发生发展的过程及机制，并为发掘CID早诊断的有效指标，探寻CID早治疗的靶点和有效方法提供一定的理论依据

糖尿病性认知障碍(CID)严重影响病人的生活质量和治疗方案的实施，但CID发病隐匿、进展缓慢，目前急需发掘CID早诊断的有效检测指标，亟待寻找CID早治疗的有效方法。海马在人和动物学习、记忆整合中起重要作用；天麻素对学习记忆能力具有一定保护作用。主要研究内容：1. 进一步深入研究大鼠CID与病程发展间的关系；2.研究CID发生发展过程中各检测因子的表达定位及水平变化，并特别关注在CID早期时出现明显变化的因子；3.早期（成模后3周）即开始使用天麻素进行干预。本项目从影响认知功能的神经递质及神经细胞信号转导的分子途径入手，运用HE染色、免疫荧光双标、Western blot、qPCR等多种实验方法，研究CID发生发展过程中，各组别大鼠海马组织的病理变化；检测多个因子的表达定位及水平变化；研究分析天麻素干预对糖尿病大鼠海马病变及各检测因子的影响。重要研究结果：1. Morris水迷宫检测结果：随病程进展，糖尿病大鼠出现不同程度的学习记忆能力下降，尤其是9周组；天麻素早期干预，历时3周后，其学习记忆能力较DM组有改善的趋势，使用6周后，改善效果较为明显。2.随病程进展，糖尿病大鼠海马组织病变逐渐加重，天麻素干预3周、6周后，可在一定程度上使DM6w+G、DM9w+G组大鼠海马组织的病变分别较DM6W+S、DM9W+S有所减轻。3.随DM病程进展，大鼠海马组织中NFкB、iNOS、eNOS、 TNFα、PKC-γ、nNOS、MMP9等因子的蛋白表达及mRNA水平较NC组分别上调，而天麻素干预则可在一定程度上使DM6w+G、DM9w+G组大鼠海马组织中上述因子的蛋白和mRNA水平分别较DM6W+S、DM9W+S组在一定程度上出现趋向正常对照组的下调。主要科学意义在于发现：1. CID与海马病变密切相关，随病程逐渐加重； 2. CID的发生发展与NFкB、PKC、nNOS、MMP9、VEGF、PKC-γ、NR2A、NR2B、 GluR-2， nNOS、iNOs、eNOS等多条神经信号通路中的多个因子密切相关；且这些因子早期就可出现明显变化，进一步深入研究后，有望从中发现早期诊断的检测指标； 3. 天麻素早期、持续较长时间干预可改善CID；天麻素改善CID的作用机制与NFкB、PKC、nNOS、MMP9等因子较为密切。