肠道菌群代谢产物氧化三甲胺与静脉血栓形成的关联及其导致易栓状态的机制研究

Trimethylamine N-oxide (TMAO) is a product of the metabolism of choline in the diet through the intestinal flora. Numerous studies have shown that elevated plasma TMAO levels increase the risk of thrombotic disease, but its impact on venous thrombosis is unclear. In the previous case-control study, our group found that plasma TMAO levels in patients with venous thrombosis were significantly higher than those in the control group. Animal experiments showed that mice with higher plasma TMAO levels had higher levels of circulating thrombin-antithrombin-complex and manifested enhanced potential of embolism in the model of venous thrombosis induced by inferior vena cava stenosis, suggesting that plasma TMAO may promote the formation of venous thrombosis through activation of the coagulation process. The current research expanded the sample size of previous study and focused on the relationship between plasma TMAO level and tissue factor activity, thrombin generation potential and microparticle tissue factor activity, as well as the impact of plasma TMAO level on venous thrombosis. The effects of TMAO on the expression and procoagulant activity of tissue factor in vitro were studied at the cellular level. Furthermore, a mouse model with high TMAO level was induced with a high choline diet and the overall tendency to thrombosis was assessed by thrombosis models induced by inferior vena cava stenosis models. This study provided new ideas for early screening, prophylaxis and individualized treatment of venous thrombosis.

氧化三甲胺（trimethylamine N-oxide，TMAO）是饮食中摄入的胆碱经肠道菌群代谢的产物。大量研究表明血浆TMAO水平升高可增加冠心病（动脉血栓）的患病风险，但其对静脉血栓形成的影响尚未明确。本课题组在前期小样本病例-对照研究中发现，静脉血栓患者血浆TMAO水平较对照人群显著升高，进一步动物的动物实验显示TMAO水平较高的小鼠具有更高的凝血酶-抗凝血酶复合物水平及更强的成栓能力，提示TMAO可能激活凝血过程并促进静脉血栓形成。本研究将进一步扩大样本量，通过Logistic回归分析对年龄、性别、制动等因素进行校正，评估血浆TMAO水平与组织因子促凝活性、组织因子微粒水平、凝血酶生成潜能等凝血指标间的相关性及其对静脉血栓形成的风险；在细胞水平及动物水平检测高血浆TMAO水平对小鼠静脉血栓形成的影响及可能的机制，为静脉血栓的早期筛查、预防及个体化治疗提供新思路。

静脉血栓栓塞症是一种严重威胁人类生存和健康的常见病、多发病，其在亚洲人群中的发病率逐年上升，约为0.3-0.57每千人。同时，静脉血栓具有较高的致死率和致残率，每年由静脉血栓导致的死亡率高达9.4%-32.3%，长期、反复发生的下肢静脉血栓易引起“血栓形成后综合征”，严重影响患者的下肢功能和生活质量，成为全球主要疾病负担之一。对具有高危血栓形成风险的个体进行早期识别有利于静脉血栓的早期防治。TMAO是红肉中所富含的胆碱、磷脂酰胆碱及左旋肉碱经体内肠道菌群的代谢所产生的小分子化合物。近年来的研究表明TMAO与冠心病、脑梗塞等动脉血栓事件的发生发展明显相关，但其对静脉血栓形成的影响尚未明确。本课题组通过建立静脉血栓样本库进行了病例对照研究，结果显示静脉血栓患者的血浆TMAO水平较对照人群有一定水平升高，将血浆TMAO&nbsp;水平按四分位数法分为四个组段（Q1：<=4.63ug/L;Q2：4.63-6.28ug/L；Q3：6.28-10.1ug/L；Q4：>=10.1ug/L），Q4段的个体患静脉血栓的风险是下四Q1的3.75倍，p=0.053。TMAO在体内的功能复杂而多样，其在整体水平对机体凝血活性及静脉血栓形成的影响尚不确切。我们以高胆碱饮食的喂养方式建立了高血浆TMAO水平的动物模型，结果显示高胆碱饮食组小鼠血浆游离TF水平较对照组无显著升高；在转录水平及蛋白水平 ，高胆碱饮食饲养的小鼠血管壁组织因子表达水平较普通饲料组小鼠均有一定水平升高 ，TFmRNA水平为对照组小鼠6.5倍（n=5，p=0.031），TF蛋白水平为对照组3.9倍（n=4 ，p=0.023）；免疫组化结果显示，与普通饲料喂养的小鼠相比，高胆碱饮食组小鼠血管壁组织因子表达更强。通过下腔静脉狭窄法诱导静脉血栓模型，从整体水平上验证了高胆碱饮食可增高血浆TMAO水平，并诱导血管壁TF的表达，从而促进静脉血栓的形成。进一步的体外实验显示，TMAO通过NF-κB及AP-1诱导了内皮细胞TF的表达。本项目从群体现象入手，以凝血途径的始动因子组织因子为切入点， 从细胞、动物、人体三个层面阐明 TMAO 水平、组织因子及血栓形成风险之间的关系，为静脉血栓的早期筛查、 预防及个体化治疗奠定理论基础。