Gut microbial metabolite trimethylamine N-oxide aggravates pulmonary hypertension

Trimethylamine N-oxide (TMAO), a metabolite derived from intestine microbial flora, enhances vascular inflammation in a variety of cardiovascular disease, and the bacterial communities associated with trimethylamine N-oxide (TMAO) metabolism is higher in pulmonary hypertension (PH) patients. The effects of TMAO on PH, however, has not been elucidated. In the present study, we found that circulating TMAO is elevated in intermediate to high-risk PH patients when compared to healthy control or low-risk PH patients. In monocrotaline-induced rat PH models, circulating TMAO is elevated; and reduction of TMAO using 3,3-dimethyl-1-butanol (DMB) significantly decreased right ventricle systolic pressure, pulmonary vascular muscularization in both monocrotaline-induced rat PH and hypoxia induced mice PH models. RNA sequencing of rat lungs on DMB revealed significant suppression of pathways involved in cytokine-cytokine receptor interaction, and cytokine and chemokine signaling. Protein-protein interaction analysis of the differentially expressed transcripts regulated by DMB showed 5 hub genes with a strong connectivity of proinflammatory cytokines and chemokines including Kng1, Cxcl1, Cxcl2, CxcL6 and Il6. In vivo, TMAO significantly increased the expression of Kng1, Cxcl1, Cxcl2, CxcL6 and Il6 in bone marrow derived macrophage. And TMAO-treated conditioned medium from macrophage increased the proliferation and migration of pulmonary artery smooth muscle cells; but TMAO treatment did not change the proliferation or migration of pulmonary artery smooth muscle cells. In conclusion, our study demonstrates that TMAO is increased in severe PH, and the reduction of TMAO using DMB reduces pulmonary vascular muscularization and alleviates PH via suppressing the macrophage production of chemokines and cytokines.氧化三甲胺（Trimethylamine N-oxide, TMAO）是一种由肠道微生物群衍生的代谢物，可在多种心血管疾病中加重血管炎症；肺动脉高压（pulmonary hypertension, PH）患者体内与TMAO代谢相关的细菌群落丰度更高。然而，TMAO对肺动脉高压的作用机制尚未阐明。本研究发现，相较于健康对照人群或低危肺动脉高压患者，中高危肺动脉高压患者的循环TMAO水平升高。在野百合碱诱导的大鼠肺动脉高压模型中，循环TMAO水平同样升高；通过3,3-二甲基-1-丁醇（3,3-dimethyl-1-butanol, DMB）降低TMAO水平，可显著降低野百合碱诱导的大鼠肺动脉高压模型以及缺氧诱导的小鼠肺动脉高压模型的右心室收缩压，并缓解肺血管肌化。对DMB干预后的大鼠肺部组织进行RNA测序分析发现，细胞因子-细胞因子受体相互作用、细胞因子及趋化因子信号通路均受到显著抑制。对DMB调控的差异表达转录本进行蛋白质相互作用分析，筛选得到5个核心基因，它们与促炎细胞因子及趋化因子（包括Kng1、Cxcl1、Cxcl2、Cxcl6及Il6）具有强连通性。体内实验表明，TMAO可显著上调骨髓来源巨噬细胞中Kng1、Cxcl1、Cxcl2、Cxcl6及Il6的表达。而经TMAO处理的巨噬细胞条件培养基可促进肺动脉平滑肌细胞的增殖与迁移；但直接用TMAO处理肺动脉平滑肌细胞，并未改变其增殖或迁移能力。综上，本研究证实严重肺动脉高压患者体内TMAO水平升高，通过DMB降低TMAO水平可通过抑制巨噬细胞分泌趋化因子与细胞因子，缓解肺血管肌化并改善肺动脉高压。