肠道菌群介导的肠源LPS参与子痫前期炎症反应机制的研究

Preeclampsia (PE) is a form of hypertensive disorder of pregnancy. Inflammation may play an important role in the pathophysiological mechanism of PE. Lipopolysaccharide (LPS) derived from gut microbial may contribute to the inflammatory responses. Pre-test of small sample showed significant difference in intestinal flora structure between PE patients and healthy pregnant women. The abundance of LPS-producing bacteria in intestinal flora of PE patients was higher than that of healthy controls. Our preliminary experiments also indicated that the fluorescence signal could be detected in the maternal-fetal interface of spontaneously hypertensive rat (SHR) in pregnancy after oral administration of FITC-LPS, and the pregnant SHR experienced PE-like syndrome such as proteinuria. These results suggest that intestinal LPS may be involved in systemic or placental inflammation during PE. But the related research has not been reported yet. This reseach intends to expand the sample size on the basis of the earlier stage. Through sequencing analysis of intestinal flora, we will clarify the relationship between LPS-producing specific flora and intestinal LPS production and expression of inflammatory factors in PE patients. We will continue to use FITC-LPS to construct PE-like rats, and to detect the effect on inflammatory reaction in rats by directional regulation of LPS-producing bacterial flora structure in rat intestine with probiotics and G- E.coli. Combination of trophoblast culture, drug inhibition experiment and SiRNA transfection experiment for TLR4 to clarify the possible signaling pathway in PE patients. This study will provide new clues for the study of molecular mechanism of PE occurrence. This study will also provide us the evidence on how to regulate the intestinal microecology and play an anti-inflammatory role purposefully. This study will lay a foundation for the prevention and control of PE.

子痫前期（PE）为妊娠期高血压疾病一种，炎症免疫反应与PE发生密切相关。肠道菌群代谢产物脂多糖（LPS）是炎症反应重要诱发因子，本项目前期小样本研究发现PE患者与健康孕妇肠道菌群结构差异显著，PE患者产LPS菌群丰度提高，利用FITC-LPS灌喂孕期高血压大鼠证实，肠源LPS可达母胎界面并诱发炎症反应和PE样蛋白尿，但PE发生时肠道菌群介导的肠源LPS是否参与炎症反应尚未见报道。本课题拟在前期基础上扩大样本量，对PE患者肠道菌群测序分析，探讨PE患者炎症反应与肠道菌群、肠源LPS产生的关系；利用LPS-FITC构建PE样大鼠，探讨通过益生菌和大肠杆菌定向调控肠道菌群对大鼠炎症反应影响；结合滋养细胞培养、药物抑制实验及针对TLR4的SiRNA转染实验，探讨肠源LPS参与炎症反应可能信号通路。为研究PE发生分子机制提供新线索，为有目的性改善肠道微生态、发挥抗炎调节作用，预防和控制PE奠定基础。

项目背景：子痫前期（Preeclampsia，PE）是以妊娠20周后出现高血压、蛋白尿等为主要临床表现的妊娠特发疾病，可致全身多脏器损伤，仍是孕产妇死亡和医源性早产的主要原因之一。由于PE发病率高居不下，其发病机制仍为国内外学者研究的热点及难点内容。过度炎症反应被认为是PE发病的重要病理基础，但影响PE发生时过度炎症反应的因素尚不完全清楚。肠道微生物代谢产物直接影响人体物质和能量代谢，同时调控肠道免疫应答，参与炎症反应和氧化应激，影响人体健康。肠道菌群失调与宿主慢性代谢性疾病如肥胖、血脂异常、胰岛素抵抗以及糖尿病等密切相关。肠道菌群对人体健康的影响被广泛认为是近年来生物医学领域重要新兴领域研究方向之一。主要研究内容：本课题通过对PE患者及健康孕妇肠道菌群变化情况，尤其是产LPS菌群比例变化情况，研究PE患者肠道微生物特征及变化规律；通过构建PE样大鼠模型，定向调控大鼠肠道微生物构成，结合LPS结合蛋白抑制剂的使用，验证产LPS菌群下调/上调后对大鼠全身或胎盘局部炎症反应的影响，进一步证实肠道菌群与PE发生时炎症反应的关联性；体外滋养细胞培养进一步证实肠源LPS参与PE发生时的炎症反应及相关信号转导通路的验证。重要研究结果：基于本项目的研究成果，发表第一标注论文6篇，其中4篇SCI，2篇核心期刊论文。关键数据及科学意义：本课题研究发现PE患者肠道菌群与健康孕妇肠道菌群存在明显结构差异，多种产LPS的G-菌属在PE患者肠道菌群中丰度较高，PE患者肠道LPS含量显著高于健康孕妇含量水平；通过FITC-LPS灌喂SHR孕鼠发现，SHR大鼠胎盘可检测到肠源LPS信号，并且LPS信号转导途径关键蛋白TLR4、MyD88以及TRIF的表达明显增强，说明肠源LPS可以诱导滋养细胞TLR4信号通路的激活，同时大鼠出现PE样蛋白尿现象。并且细胞实验证实核心炎症因子IL-1β、IL-6、TNF-α以及MCP-1表达水平明显高于对照组，而IL-10含量明显低于对照组，与已有报道PE大鼠模型炎症因子表达情况类似，进一步说明肠源LPS可影响PE发生时胎盘界面局部炎症反应。