艾纳香油及其活性成分对LPS致鸡血管内皮细胞炎症信号通路关键靶向影响的研究

Blumea balsanifera DC. is a characteristic resource in Guizhou. However, there are only few literatures about its active constituent of anti-inflammatory pharmacological evaluation, let alone an independent intellectual property rights. Nowadays, both its anti-inflammatory mechanism and target are not clear, the research and application about prevention and control of inflammatory diseases in animals is less. Therefore, in this project, we intend to take volatile oil and its active constituent for research objects, screen anti-inflammatory compounds through analysis of experiments by GC-MS, evaluate the effect of volatile oil and its main active constituent on inflammation in chiken induced by LPS through Changes of clinical symptoms, related indexes of animals and pathological changes, and study the influence of volatile oil of the Blumea balsamifera DC. and its active constituent on key target of inflammatory signaling pathways and cytokines secretion in chicken vascular endothelial cell by siRNA，qPCR，ELISA，Western Blot. Hoping implementation of this project will help to reveal specified anti-inflammatory compounds and its regulatory mechanism on inflammation in chiken induced by LPS and key target gene of inflammatory signaling pathways in activated chicken vascular endothelial cell, and to further exploite and find new anti-inflammatory compounds of Blumea balsamifera DC.. Which contributes to lay a solid scientific basis for the further development and utilization of medicinal resource Blumea balsamifera DC., establish the foundation for future development and application of volatile oil in animal disease areas, and afford a new way to develop characteristic economy of Guizhou.

艾纳香为贵州特色资源，但其独立自主知识产权的活性成分抗炎药理评价研究报道极少，其作用机制、作用靶向不明确，在动物炎症性疾病的防控方面研究及应用甚少。为此，本项目拟以艾纳香油及其活性成分为研究对象，通过GC-MS联用、体内外实验分析筛选抗炎物质，从临床症状及相关指标、病理变化等评价其抗LPS致雏鸡炎症的作用，以血管内皮细胞为靶向，采用siRNA、qPCR、ELISA、Western Blot等方法综合分析其对细胞分泌炎性因子作用以及细胞炎症信号通路关键靶基因的影响。通过项目实施可揭示艾纳香油抗炎物质不清晰，以血管内皮细胞中关键基因为靶向阐明艾纳香油及抗炎物质对LPS致炎雏鸡的影响机制，深度挖掘和发现艾纳香油中新的抗炎物质，为进一步开发利用艾纳香的药用特色资源打下坚实的科学依据，为艾纳香油在动物疾病方面应用的研发打基础，为贵州省特色经济发展提供坚实的科学基础。

课题组进行4个方面研究：艾纳香油（BBO）抗炎物质的研究，BBO及其活性物质抗 LPS 致雏鸡炎症的研究，BBO及其活性物质对 LPS 致细胞分泌炎症因子及机制的研究，BBO及其活性物质对 LPS 致血管内皮细胞炎症信号通路关键靶向的研究。.结果显示：不同产源BBO含有一定的共有成分，且相对含量不同，建立了同时检测BBO中几个化合物的HPLC方法，研究了质量标准；筛选出β-石竹烯（BCP）、(-)-芳樟醇能降低实验性炎症发生，BBO对金黄色葡萄球菌MIC值9.77 μg·mL-1；很难建立稳定的LPS致雏鸡炎症模型，BBO、BCP、(-)-芳樟醇、(-)-龙脑能提高CCL4联合LPS致雏鸡存活率；BBO、BCP、(-)-芳樟醇能扭转 LPS 导致的细胞死亡以及形态变化，降低LPS所致细胞促炎因子、炎症介质和粘附分子的分泌表达，能降低LPS所致花生四烯酸（AA）途径、Nrf2/HO-1 通路、TLR4通路及NF-κB通路中相关因子表达水平，阻止 p65 核位移；LPS作用鸡血管内皮细胞有10个蛋白差异达1.5倍，其中THBS1对细胞促炎因子TNF-α、IL-6、IL-1β和TGF-β1的表达有影响，BBO和BCP能通过抑制LPS介导的THBS1、ACSL1、ACSL4、TIMP3的表达。BCP作用于LPS致炎的人血管内皮细胞有63个蛋白上调77个蛋白下调，其中SOS1、GEM、Akirin2、Scribble与炎症相关，SOS1、Akirin2与NF-κB信号通路存在相关性；与TLR4信号通路关键蛋白分子虚拟筛选有作用的成分有：艾纳香烯A、香橙烯、α-芹子烯、BCP等。.通过项目实施，明确了BCP、(-)-芳樟醇是BBO抗炎物质，LPS致炎雏鸡的模型有待进一步探讨，BBO及BCP、(-)-芳樟醇通过影响AA途径、Nrf2/HO-1 通路、TLR4及NF-κB中相关因子的表达，阻止NF-κB的 p65 核位移，降低细胞促炎因子、炎症介质和细胞粘附分子分泌发挥抗炎作用，扭转 LPS 导致细胞的死亡及形态变化。发现THBS1（鸡）在炎症中起作用，SOS1、Akirin2（人）等与炎症相关，且BBO和BCP对其有影响，这可能是抗炎新靶点。.以上结果为进一步开发利用艾纳香的药用特色资源打下坚实的科学依据，为BBO在动物疾病方面应用打基础，为贵州省特色经济发展提供坚实的科学基础。