肠菌紊乱促进卒中脑损伤：临床关联分析及菌群移植动物模型研究

Gut microbiota has newly become a common risk factor of many important diseases, but it’s role in post-stroke brain damage is still unknown. Our research group recently found that the gut microbiome of acute stroke patients showed obvious dysbiosis. Besides, a linear regression was found between their gut microbial index and brain damage index NIHSS score. Our present project assumes that: the degree of disorder of post-stroke gut microbiota is affected by pre-stroke gut microbiota characteristics and stroke events. Post-stroke pathological gut microbiota promotes inflammation, inhibition of angiogenesis, thus increasing acute ischemic brain injury. To check this assumption, our project plans to expand the subject sample. Using high-throughput sequencing 16S rRNA tag sequencing and random forest analysis, we plan to build a microbial index of stroke patients and verify their statistical correlation with brain injury. Secondly, by fecal microbiota transplantation, we plan to examine the impact of different pre-stroke gut microbiota in asymptomatic group and two different infarction modeling technologies on post-stroke gut microbiota disorder. Next, according to the microbial index, we’ll choose different fecal bacteria samples from acute stroke patients, and then transplanted them into C57 mice. After mild infarction modeling, we verify the Th cell polarization, angiogenesis stimulated by VEGF, infarct size and neurological behavior indicators. Last but not least, we transplant the healthy gut microbiota into mice with severe infarction modeling to reverse the dysbiosis. We also will examine the impact of acute gut microbiota improve on stroke prognosis. Our research is expected to verify a new mechanism of acute stroke damage. Meanwhile, we hope to develop the gut microbiota as a new stroke risk assessment factor and an intervention target.

肠道菌群是新发现的多疾病共同风险因子，但其对卒中急性期病理影响未明。课题组新近发现，急性卒中患者肠菌明显失调。并且，患者肠菌紊乱指数与脑损伤指标NIHSS评分线性相关，提示二者可能具有内在联系。本课题假设：卒中后肠菌紊乱程度受卒中前肠菌特征以及卒中事件共同影响；卒中后病态肠菌促进炎症反应，抑制血管再生，从而增强急性期脑损伤。课题拟扩大样本，通过16S高通量测序及随机森林法，建立卒中患者肠菌紊乱特征谱指数，验证其与脑损伤统计相关性；通过粪菌移植技术，检测轻重两种梗死造模技术以及无急性事件人群不同初始肠菌对卒中后肠菌紊乱的影响；移植急性期患者不同紊乱程度粪菌，轻度梗死造模，分别检测Th细胞极化，VEGF刺激下的血管再生，梗死面积及神经行为学等指标；最后，对重度梗死造模小鼠移植健康粪菌，检验急性期肠菌改善对卒中预后的影响。本研究有望探明卒中急性期损伤新机制，建立新的肠菌卒中风险评价及干预靶标。

研究背景：肠道菌群是新发现的多疾病共同风险因子，但其对卒中急性期病理影响未明。本项目旨在研究肠道菌群是否对卒中后损伤发挥重要作用。.研究内容：本课题组拟研究急性缺血性脑卒中（acute ischemic stroke, AIS）患者肠菌紊乱特征谱，建立相对稳定的卒中肠菌紊乱指数（stroke dysbiosis index, SDI），确定急性期患者肠菌紊乱与脑损伤关键指标存在统计相关性；进一步采用粪菌移植实验，将高低指数两组患者的粪便菌群移植到C57BL/6小鼠，然后对小鼠进行大脑中动脉梗阻（Middle cerebral artery occlusion, MCAO）造模，观察小鼠术后脑损伤程度；通过流式实验检测小鼠术后外周T细胞亚群分化程度，分析菌群失调与脑损伤和免疫之间的关联，进一步探明急性期肠菌紊乱是否对卒中后损伤具有重要作用。.重要结果：经过年龄匹配，本课题组建立一个194人的卒中病例对照队列，包括104例急性期缺血性卒中患者和90例健康对照人群，通过肠菌分析，我们找到18种卒中患者相关的主要菌属，探索性建立一个诊断卒中精确性达到74.9%的卒中肠菌失调指数SDI模型，指数高低代表卒中患者肠菌失调程度；SDI模型在验证队列（153人，83例卒中患者和70例健康对照）的ROC曲线结果为AUC 84.3%；SDI与脑损伤（NIHSS，r=0.208，P=0.034）、临床结局（mRS，r=0.281, P=0.004）正相关，多元逻辑回归分析结果发现SDI是重度卒中（NIHSS≥8） (OR: 1.019, 95%CI: 1.004–1.034) 和早期不良结局 （mRS>2）（OR: 1.022, 95%CI: 1.008–1.035)的独立预测因子。粪菌移植动物实验结果显示：卒中患者紊乱程度越高的菌群，移植后导致受菌小鼠的脑损伤程度的加重，伴外周IL-17+γδ T 细胞的增高，而具有抑制IL-17+γδ T 细胞分化的Treg细胞的减少。.科学意义：本课题组建立的卒中肠菌失调指数模型，是一个较好的评估卒中患者肠菌失调程度的指标。该指数与患者的预后显著相关，并与卒中小鼠模型的预后有因果关系。我们的SDI模型增加了肠道菌群与卒中之间的定量证据，表明肠道菌群在卒中的潜在临床应用价值。