從血清學和口腔微生物群之角度研究阿滋海默症與牙周病之間的關連：病例對照研究

Abstract

研究背景：阿茲海默症是一種神經退化性疾病，致病機轉被認為和中樞以及周邊發炎反應有關係。牙周病亦是一種與發炎高度相關的疾病，流行病學研究指出牙周病患者有較高的風險發展成阿茲海默症，但牙周病如何影響阿茲海默症的機轉尚未釐清。本研究藉由觀察阿茲海默症患者與對照者(非阿茲海默症患者)之臨床牙周健康狀況、血清相關發炎因子濃度、唾液細菌種類分布，以比較阿茲海默症患者的失智嚴重度(CDR)、血清標記因子、口腔菌系組成與牙周病嚴重程度之間的關係，以探討阿茲海默症及牙周病之間的相關性。 材料與方法：共31名輕度至中度之單純阿茲海默型失智患者(AD)與31名健康對照組(Control)納入本實驗，進行問卷填寫以調查人口學資料、個人習慣、過去病史、家族病史、牙科照護習慣，並且進行全口牙周檢查（殘留齒數、牙周囊袋深度、臨床附連喪失、牙菌斑指數、牙齦發炎指數、放射線骨高度），並檢測血清中A42、pTau、Tau、IL-10、TNF-α、IL-1β、IL-6、IL-8、hsCRP、anti-P. gingivalis LPS抗體的濃度。亦收集受試者的唾液進行DNA萃取、16S rDNA (V3-V4) 次世代基因定序(Illumina®)以分析口腔微生物菌系組成。 結果：1. 首先以年齡-性別配對方法篩選後得到兩組各20名受試者：AD組的血清標記因子Tau、hsCRP、Anti-P. gingivalis LPS抗體濃度顯著高於Control組，Anti-P. gingivalis LPS抗體和hsCRP之濃度呈顯著正相關，和IL-10之濃度呈顯著負相關；hsCRP之濃度則和A42、Tau、pTau之濃度呈顯著正相關，和IL-10之濃度、後牙植牙數量呈顯著負相關。此外，AD組的教育程度顯著低於Control組、口腔衛生照護習慣亦顯著不足，但並未發現AD組之牙周疾病嚴重度、缺牙情況、系統性疾病複雜程度相較Control組嚴重。2. 其次，比較所有收案AD組(n=31)：臨床附連喪失小於4釐米(CAL<4mm) 和CDR分數之間為顯著負相關，血清中的A42濃度和Tau、pTau、hsCRP、IL-1濃度呈正相關。3. 最後比較所有收案之AD組(n=31)和Control組(n=31)之口腔微生物菌系差異：兩組間並無物種豐度和分佈比例上的顯著差異，唯有5~6%的物種組成差異，若以菌屬劃分發現Prevotella、Peptococcus 、Faecalibacterium、Anoxybacillus在AD族群中顯著較多，若以菌種劃分則發現Prevotella buccae 在AD組中顯著較多。 結論：本研究發現失智越嚴重，臨床牙周附連喪失程度越多。AD組的血清hsCRP濃度顯著較高，與AD成因相關之Tau蛋白濃度亦較高，顯示慢性發炎和AD的正關聯性，且Anti-P. gingivalis LPS抗體和hsCRP濃度呈正相關，提供牙周病和系統性慢性發炎的相關性。但AD組和Control組的牙周病嚴重度和口腔微生物菌系組成不具顯著差異。雖發現AD族群口腔微生物菌系中有極少量細菌比例顯著較多，但這些細菌對於AD可能造成的影響仍須後續更多的研究探討。

Objectives: Alzheimer disease (AD) is a neurodegenerative disease associating with central and peripheral inflammatory responses. Periodontal disease is a bacteria-induced chronic inflammatory disease. Epidemiological studies have reported that patients with periodontal disease have a higher risk of developing AD, but the mechanism was not yet been fully understood. The aim of this study was to explore the association between AD and periodontal diseases in a case-control study. Methods: 31 patients with slight or moderate AD (no mixed type dementia) and 31 controls without AD were enrolled in this study. Each participant or the main care giver was asked to fill the questionnaire about the individual’s family history of dementia, family history of periodontal disease, present and past medication history. In addition, past dental maintenance frequency, daily oral hygiene care and personal habit such as smoking were recorded. All the participants received periodontal charting and radiographic film to record periodontal parameters, the number of remaining teeth and the bone level of bilateral mandibular posterior teeth. In addition, the blood was collected to detect serum biomarker including A42, Tau, pTau, pro-inflammatory cytokines, anti-P. gingivalis LPS antibodies, and triglyceride. The saliva samples were also collected for analyzing the oral miciobiota by 16S rDNA (V3-V4) NGS technique. Results: After the age and gender matching (AD/control = 20/20), the AD patients showed lower education level, and higher serum concentration of Tau、hsCRP、anti-P. gingivalis LPS antibody with significancy. In addition, serum anti-P. gingivalis LPS antibody was positively correlated to serum hsCRP, but negatively correlated to serum IL-10. Also, serum hsCRP was positively correlated to serum A42, Tau and pTau; but negatively correlated to serum IL-10 and number of posterior dental implants. However, there was no significant difference between groups in the severity of periodontal disease and the complexicity of systemic medical history. As for intra-group analysis of all AD participants (n=31), CDR score was negatively correlated to clinical attachment loss (CAL)<4mm with significancy. Besides, serum concentration of A42 was positively correlated to Tau、pTau、hsCRP、IL-1. Finally, the main composition of the oral microbiota was similar with only 5~6% difference in bacteria diversity between two groups (AD/control = 31/31). Four bacteria genus (Prevotella, Peptococcus, Faecalibacterium, Anoxybacillus) and 1 bacteria species (Prevotella buccae) were signigicantly associated with AD. Conclusions: With the limitation of this study, we conclude that AD was associated with higher serum hsCRP, Tau protein, and anti-P. gingivalis LPS antibod. However, the periodontal condition of in AD group was not significantly severer then the control. Within the AD patients, the severity of the dementia was positively correlated to the amount of periodontal clinical attachment loss. Even though very few bacteria abundance of oral micriobiota was significantly higher in the AD group, the majority of the composition was similar in the two groups. Further studies are warranted to varify the role of these bacteria in the pathogenesis of AD.