Table_1_Experimental Periodontitis Deteriorated Atherosclerosis Associated With Trimethylamine N-Oxide Metabolism in Mice.docx

BackgroundPeriodontitis is considered a risk factor for atherosclerosis, but the mechanism is not clear. It was reported that oral administration of Porphyromonas gingivalis altered the gut microbiota in mice. Gut dysbiosis and the intestinal metabolite trimethylamine N-oxide (TMAO) were verified to be associated with atherosclerosis. Therefore, the possible TMAO-related mechanism between periodontitis and atherosclerosis needs to be explored.
MethodsExperimental periodontitis was established by oral administration of P. gingivalis for 2 months in ApoE−/− mice. Mouse hemi-mandibles were scanned using Micro-CT. Quantification of TMAO was performed using liquid chromatography–tandem mass spectrometry. Mouse feces were collected and the bacterial DNA was extracted, then the gut microbiota was analyzed using 16S rRNA genes. Atherosclerotic lesion areas were quantified. Livers, small intestines, and large intestines were analyzed for gene expression.
ResultsAggravated atherosclerosis plaques were found in experimental periodontitis mice. Plasma TMAO, a pathogenic factor of atherosclerosis, was initially found to be increased in periodontitis mice. Changes in the composition and abundance of the intestinal microflora of periodontitis mice were found. Flavin monooxygenase 3 (FMO3), the catalyzing enzyme of TMAO in the liver, was significantly increased, accompanied by an increase of IL-6 in liver, the abnormal intestinal integrity and enhanced plasma LPS. The IL-6 and LPS were verified to be able to increase FMO3 in HepG2 cells.
ConclusionOur research discovered that experimental periodontitis in ApoE−/− mice induced gut dysbiosis and an increase in TMAO. These results suggest a possible mechanism by which periodontitis may accelerate atherosclerosis by influencing the intestinal microbes and the metabolism, which were triggered by inflammation of the liver and intestine.

【背景】牙周炎（Periodontitis）被认为是动脉粥样硬化（atherosclerosis）的潜在危险因素，但其具体致病机制尚未明确。已有研究显示，口服牙龈卟啉单胞菌（Porphyromonas gingivalis）可改变小鼠肠道菌群（gut microbiota）组成。肠道菌群失调（gut dysbiosis）与肠道代谢产物氧化三甲胺（trimethylamine N-oxide, TMAO）已被证实与动脉粥样硬化密切相关，因此牙周炎与动脉粥样硬化之间潜在的TMAO介导机制有待进一步探究。
【方法】本研究对ApoE基因敲除（ApoE−/−）小鼠口服牙龈卟啉单胞菌，构建为期2个月的实验性牙周炎模型。采用显微计算机断层扫描（Micro-CT）对小鼠半侧下颌骨进行扫描成像；通过液相色谱-串联质谱（liquid chromatography–tandem mass spectrometry）定量检测血浆TMAO水平；收集小鼠粪便样本，提取细菌基因组DNA，利用16S核糖体RNA（16S rRNA）基因测序技术分析肠道菌群组成；对动脉粥样硬化斑块面积进行定量检测，并对肝脏、小肠及大肠的基因表达水平进行分析。
【结果】实验性牙周炎小鼠的动脉粥样硬化斑块负荷显著加重。本研究首次发现，牙周炎小鼠血浆中作为动脉粥样硬化致病因子的TMAO水平显著升高；同时，牙周炎小鼠的肠道菌群组成及丰度发生明显改变。肝脏中催化TMAO合成的黄素单加氧酶3（Flavin monooxygenase 3, FMO3）表达量显著上调，伴随白细胞介素6（IL-6）水平升高、肠道屏障功能异常及血浆脂多糖（lipopolysaccharide, LPS）水平升高。进一步细胞实验证实，IL-6与LPS均可在HepG2细胞中上调FMO3的表达。
【结论】本研究证实，ApoE基因敲除（ApoE−/−）小鼠的实验性牙周炎可诱导肠道菌群失调并升高血浆TMAO水平。上述结果提示，牙周炎可能通过影响肠道菌群及其代谢通路，介导肝脏与肠道炎症反应，进而加速动脉粥样硬化进展，该发现为二者的潜在关联提供了全新的机制阐释方向。