慢性牙周炎致老年认知功能障碍机制的研究

Chronic periodontitis (CP) is the most common oral chronic infectious disease. According to the third national oral health epidemiological investigation，the incidence of CP is up to more than 80% of adult population，it is well discussed the positive linking of CP and systemic diseases in the dental science researches, and CP has been accepted as an independent risk factor of diabetes and cardiovascular diseases. Recent years，the correlation between CP and brain has also been spotlighted，however，the linking of CP with age-dependent cognitive impairment and its cellular and molecular mechanisms are still unknown. Basically on localizing in the comprehensive university which covers medical and dental sciences as well as the long-lasting collaboration with researchers aboard in dental science，our research group rises a project to clarify the possibility of CP positive linking to the aging-dependent cognitive impairments as well as its cellular and molecular mechanisms. Firstly，we will clarify the relevance between the clinic indexes and age-dependent cognitive impatient in CP patients using clinical epidemiological analysis. Secondly，we will determine the evidence of cognitive impatient and the cellular mechanism in CP mice models using electrical physiology technologies. Thirdly，we will analyze the dynamically systemic inflammation changes in CP patients and CP mice using flow cytometer and immunohistochemical technologies. Fourthly，we will analyze the microglia-mediated neuroinflammation and the neuron damages in the brain of CP mice using genomics and proteomics technologies such as QRT-PCR，western blot as well as ELISA. Finally，we will clarify the molecular mechanisms using the cultured microglia and neuron to further confirm the evidence in vivo experiments. We believe that our projects will give a new idea from dental science viewpoint for prevent the cognitive impairments with aging and the aging-related neurodegeneration diseases such as Alzheimer’s disease.

慢性牙周炎与全身系统性疾病密切相关，其对脑的影响日益受到关注。我们前期研究发现牙龈卟啉单胞菌（P.g）及其主要毒力因子脂多糖(P.g LPS)可上调小鼠脑内小胶质细胞释放促炎因子，并发现慢性关节炎中年大鼠小胶质细胞释放促炎因子可诱导记忆功能低下。为了解明P.g及P.g LPS致老年认知功能障碍的机制，本实验采用分析不同年龄慢性牙周炎患者认知功能的表现，探讨人慢性牙周炎与老年认知功能的关系；长时程增强效应和水迷宫实验评价慢性牙周炎小鼠增龄性认知功能低下的变化；流式细胞术、免疫组织化学技术检测人及牙周炎小鼠炎症的动态；QRT-PCR，Western blot等技术分析牙周炎小鼠诱发脑小胶质细胞活化依赖性脑炎症；CKK8法和流式细胞术分析神经元细胞的损伤情况。以阐明慢性牙周炎与老年认知功能障碍的相关性及其机制，从口腔医学视点为预防老年及阿尔茨海默病等神经退行性认知功能障碍开辟新的思路。

近年来，慢性牙周炎与老年认知功能障碍的联系已逐渐在学术界内得到公认，但其内在机制还不甚明了。为进一步探究慢性牙周炎致老年认知功能障碍的机制，依据项目计划书中的研究内容，课题组采用临床流行病学病例对照，评价了老年慢性牙周炎患者的认知能力，确认了慢性牙周炎患者认知功能的降低。我们使用流式细胞术和ELISA检测了患者的外周血，发现了单核/T细胞数量的增加以及促炎因子TNF-α的上调，这证明了牙周炎患者的外周系统处于一种慢性炎症状态之中，且产生了阿兹海默患者的脑内标志性物质。体内牙周炎的动物模型实验中，我们检测到中年牙周炎小鼠脑、肝、脾等组织中促炎因子、Cat B、AβPP等物质表达水平的上调，Morris水迷宫法确认了其认知功能的下降，这进一步证明了年龄与牙周炎诱导认知障碍间的联系。为了进一步阐明其中机制，我们进行了体外实验。我们发现P. g及P. g LPS可以促使小鼠巨噬细胞激活并表达炎症相关产物（TNF-α、IL-1β、IL-6及特异性受体TLR2）以及阿兹海默病相关物质（AβPP、CatB）。这种被激活的巨噬细胞能够通过促进软脑膜JAK2、p38蛋白的磷酸化来介导促炎因子的上调，并进一步引发小胶质细胞的炎性应答。小胶质细胞释放的炎症信号，可以引发神经元细胞的损伤，从而损害神经系统。C5aR拮抗剂（PMX205）和天然植物提取物（松柏醛）可分别抑制巨噬细胞和软脑膜细胞的炎症反应，从而阻断炎症信号在细胞间的传递。本实验从临床研究、体外实验、体内实验三个方面阐明了慢性牙周炎对老年认知功能障碍的促进作用及机制，从口腔医学视点为预防阿兹海默等认知功能障碍疾病开辟了新的思路。