芳樟醇干预巴氏杆菌感染过程中Nrf2相关信号传导通路的研究

Linalool is an effective component of traditional Chinese medicine honeysuckle. In our previous research, it has been shown that linalool has significant antibacterial activity against Pasteurella and anti-inflammatory role by using our signal transduction pathway platform. But its anti-inflammatory mechanism of action in the bacterial infections is not clear. Aiming at this problem of LPS induced intense inflammatory response that goes against the rehabilitation of the disease with Pasteurella induced infection, in this project, with the molecular biological and modern immunological related techniques, starting from Nrf2 signaling pathways, the protective effect and regulatory impact analysis in Nrf2-Keap1, Nrf2-associated signal transduction pathway such as NF-κB and MAPKS of linalool to the pathological models with Pasteurella multocida induced infection and LPS-induced sepsis will be attempted to detect using Nrf2 knockout(Nrf2(-/-)) and Nrf2 wild-type(+/+) mice; Nrf2-Keap1, Nrf2-associated signal transduction pathway such as NF-κB and MAPKS will be analyzed in vitro with Western Blot, Real-time PCR, electrophoretic mobility shift assay(EMSA), and cyto-immunofluorescence staining technique for determining key knots in the regulating the inflammatory response, searching for the pathway and some new drug targets by the action of linalool, attempting to further elucidate the molecular mechanism of linalool's anti-inflammatory properties. The research consequences may offer theoretical guidance and experimental reference for the treatment of the inflammation with Pasteurella induced infection. It may lay the foundation for the further exploitation and application of the linalool as a lead compound and for the new drug targets screening.

芳樟醇是中药金银花的有效成分，我们前期研究发现，芳樟醇具有显著的抗巴氏杆菌活性，通过课题组建立的信号传导通路研究平台发现芳樟醇抗炎作用显著，但在感染性疾病中其抗炎作用机制尚不明确。因此针对巴氏杆菌感染时LPS导致剧烈的炎症反应而使疾病恶化，本项目拟应用分子生物学和现代免疫学相关技术，从Nrf2信号传导通路入手，评价芳樟醇在Nrf2野生型、基因缺失型小鼠中构建的多杀性巴氏杆菌感染和LPS诱导的败血症模型中的保护作用；并采用Western免疫印迹、Real-time PCR、EMSA、免疫荧光等方法，检测芳樟醇对Nrf2-Keap1及Nrf2相关的NF-κB和MAPKS等信号传导通路的影响，确定调控炎症反应的关键节点，寻找芳樟醇作用的Pathway和新的药物靶标，深入阐明芳樟醇抗炎的分子机制，为芳樟醇作为先导化合物进一步开发应用研究及新药靶标的筛选奠定基础。

芳樟醇（linalool）是从芳香植物中提取出来的天然精油类物质，具有抗炎、镇痛等多种生物活性。多杀性巴氏杆菌(Pasteurella multocida,Pm)能够引起畜禽等多种动物感染发病，多杀性巴氏杆菌病例以急性败血症和肺脏炎症为临床特征。核因子相关因子2(nuclear factor (erythroid-derived 2)- related factor2, Nrf2)是细胞抗炎、抗免疫和抗氧化应激反应的重要调节因素。在细菌感染性疾病中基于Nrf2信号通路的具有抗炎作用的中药单体少有研究。我们利用细胞生物学和分子生物学技术，进行了芳樟醇干预巴氏杆菌感染和非感染过程中Nrf2相关信号传导通路的研究。研究发现芳樟醇能够抑制LPS和多杀性巴氏杆菌诱导的细胞内的TNF-α 和IL-6 的表达，能抑制NF-kB和MAPK通路的激活，表明芳樟醇在体内外具有明显的抗炎活性。我们应用EMSA来测定芳樟醇对Nrf2蛋白DNA结合活性的影响。芳樟醇作用后显著提高了Nrf2蛋白DNA结合活性，并呈剂量依赖性增加。芳樟醇处理肺泡上皮细胞后，检测Nrf2从胞浆到细胞核显著增加，呈现剂量依赖关系。Nrf2的下游分子HO-1的表达也呈显著增加。表明芳樟醇可以对抗呼吸道上皮细胞的氧化损伤。通过Nrf2siRNA干扰发现Nrf2能够参与到多杀性巴氏杆菌诱导的炎症中，并且芳樟醇的治疗作用也很大部分依赖于对Nrf2的激活。芳樟醇可以降低小鼠肺脏多杀性巴氏杆菌定殖数、MPO活性以及炎性细胞因子TNF-α和IL-6的含量；芳樟醇可以有效保护小鼠多杀性巴氏杆菌肺炎，降低肺炎小鼠的死亡率；缓解小鼠肺水肿和肺损伤症状；增加肺泡灌洗液中SOD的水平，降低ROS的含量。以上研究结果提示芳樟醇通过激活Nrf2可潜在的用于多杀性巴氏杆菌肺炎的治疗。