基于“肠-肝轴”的乌药抗慢性酒精性肝损伤的作用机制研究

As we know, intestinal endotoxemia (IETM) was the key pathogenic factor of alcoholic liver injury (ALI) and gastrointestinal disorder, intestinal flora maladjustment and high intestinal permeability induced by alcohol was the onset of IETM. As results of previous research show, Lindera aggregata (Wuyao in Chinese name, WY) has good protective effect on ALI, but the mechanism of WY on anti ALI was unrevealed. Previous researchs show that WY could siginificantly reduced the level of inflammatory factors in serum and LPS in portal vein, improve pathological changes of small intestine, downregulate the expressions of TLR4 et al.,. According to the effects of WY on regulating gastrointestinal function and anti-microbial, we deduced that WY could protect liver from alcoholic injury by regulating gastrointestinal function, protect intestinal mucosal barrier, allevialte IETM and inhabit inflammatory reaction induced by LPS-TLR4 signal pathway. As stated above, methods such as Elisa, 16S rRNA gene sequencing, RT-PCR and Western-blot and rats with alcololic liver injury were carried out to observe the effects of WY on anti alcoholic liver injury and to illuminate the relationship between gastrointestinal function regulating and ALI protection. Caco-2 and RAW264.7 cell models were used to observe the influence of WY on expression of tight junction associated protein and activation of inflammatory signal pathway mediated by TLR4, and to reveal the mechanism of WY on improving intestinal permeability and anti inflammation.

已知肠源性内毒素血症(IETM)是导致酒精性肝损伤(ALI)的关键因素，而酒精引发的胃肠道功能紊乱、肠道通透性增加是形成IETM的始动原因。现有研究证实，乌药对急性ALI具有良好的保护作用，但具体作用机制不清。前期研究结果显示，乌药能降低血清炎症因子和门静脉LPS水平，改善小肠病理学改变，下调肝脏TLR4等的表达；结合乌药调节胃肠道功能、抗菌等作用，推断乌药可能通过调节肠道功能，保护肠粘膜屏障，减轻IETM，阻断LPS-TLR4介导的炎症级联反应而保护酒精性肝损伤。拟采用ELISA、RT-PCR及Western blot等方法，明确乌药对肠道功能、肠黏膜屏障及ALI的保护作用，阐述乌药调节肠道功能和保护ALI作用的相关性，阐明其抗ALI作用机制；通过体外Caco-2、RAW264.7细胞模型，观察乌药对紧密连接蛋白表达以及TLR4介导的炎症通路的影响，阐明其改善肠道通透性及抗炎作用机制。

酒精性肝损伤（ALI）中存在异常的“肠-肝轴”，乌药为传统理气中药，对肠道功能具有良好的调节作用和抗ALI作用，但机制不明。本项目旨在明确乌药调节肠道功能与其抗ALI间的相关性，阐述其改善肠道通透性及抗炎作用机制。整体动物实验，开展了乌药醇提取物（LREE）抗ALI量效关系研究，结果发现，在0.2~2 g/kg剂量范围，乌药改善肠道功能，降低血清炎症因子水平及保护ALI的作用较好。LREE对急、慢性ALI模型大鼠的作用及机制研究发现，LREE能改善模型大鼠血清肝功能指标，改善脂代谢异常，增强抗氧化能力；能调节ALI大鼠血清二胺氧化酶和胃泌素水平，改善肠道通透性；能调节模型大鼠肠道菌群结构，增加Ruminococcus和Lactobacillus比例；上调小肠紧密连接蛋白claudin-1和occludin的表达水平，改善酒精诱导的肠粘膜微绒毛结构损伤；能有效降低模型大鼠血清IL-8、TNF-α等炎性因子水平及门静脉血内毒素水平，减轻肠源性内毒素血症；能下调肝组织TLR4、CD68等蛋白表达水平，改善肝组织病理学改变。LREE对2,4,6-三硝基苯磺酸（TNBS）诱导的大鼠肠炎模型作用研究结果发现，LREE能降低模型动物肠指数和单位长度肠重，降低肠道病理评分；能增加肠系膜淋巴结T淋巴细胞数，降低Th比例，增加Tc比例，上调FoxP3/Th比值，改善肠道免疫亢进；能抑制结肠组织COX2表达，减轻肠道炎症反应，对TNBS诱导的肠炎具有良好的拮抗作用。体外细胞实验，建立了LPS诱导的RAW264.7体外炎症细胞模型，确定了LREE对RAW264.7细胞的IC50、IC5、IC1浓度，发现LREE能有效抑制RAW264.7炎症细胞释放IL-6、MCP-1等炎症因子，拮抗炎症反应；初步确定了LREE对caco-2细胞的浓度范围。项目的研究，进一步明确了乌药通过改善肠道功能从而拮抗ALI的作用，初步阐明了可能的作用机制；提示“肠-肝轴””可作为酒精性肝病防治的重要切入点。项目的研究可赋予中药传统理气功能新的科学内涵，促进乌药防治酒精性肝损伤及肠道疾病相关产品的研发，促进乌药产业的发展。研究结果已发表论文10篇，其中SCI 2篇；申请国家发明专利2项；获浙江省医药卫生科技三等奖1项；培养硕士研究生2名。