海洋荔枝螺基于LPS/TLR4信号通路抗炎生物碱的研究

Thais Roeding，as one of the most popular marine foods in Fujian Province, is proved with a strong anti-inflammatory effect according to the traditional Chinese medicine records and long-term using among local people. However, there are few public reports about the active components from this snail due to the restriction of regionally distribution. For this reason, it is very significant to find the anti-inflammatory components and study their molecular action mechanism at the cellular level. In this project, we will isolate and screen the anti-inflammatory alkaloids from Thais Roeding under the bioactivity assay guidance through bee venom PLA2 enzyme inhibition, NF-kB, AP-1 report gene and nuclear p65 observation. The active alkaloids are further purified by the column chromatography methods and identified of their chemical structures by means of the spectroscopy analysis such as FTIR, ESI-MS and NMR. Finally, we will find the effects of the purified alkaloids on the DNA transcription and translation of some important vascular cell adhesion molecules, chemokines factors and inflammatory factors involved in LPS/TLR4 signal pathway within human umbilical vein endothelial cell line and mouse leukaemic monocyte macrophage cell line. At the same time, the effects of the alkaloids on the adhesion between vascular cells HUVEC and inflammatory cells HTP-1 will be studied. Based on these studies, the possibe molecular anti-inflammatory mechanism of the active alkaloids from Thais Roeding is expected to be proposed.

荔枝螺是福建海域常见食用螺类，中药古方记载和民间长期食用证实具有良好的消炎止肿作用，但由于自然分布的地域性，国内外对其生物活性物质的研究还较少，因此对荔枝螺抗炎药效物质基础和作用机制的探究具有重要意义。本项目拟以福建海域荔枝螺为研究材料，通过蜂毒磷脂酶抑制，NF-kB、AP-1 报告基因激活以及p65 入核观察等手段指导分离对LPS/TLR4 信号通路具抑制作用的抗炎生物碱成分，并对该活性生物碱类化合物单体或组分进行纯化和结构鉴定。进而在人血管内皮细胞系和小鼠单核巨噬细胞系中，研究荔枝螺活性生物碱对LPS/TLR4 信号通路中主要炎症因子转录和表达水平的调节作用，以及对血管细胞与炎性细胞粘附作用的影响，力求阐明生物碱的抗炎机制。通过发现荔枝螺中具抗炎药效的物质基础，分析该组分在血管细胞内对炎症信号通路的影响，为筛选高效抗炎药物，理解炎症发生机制提供理论支持。

荔枝螺是福建海域常见食用螺类，中药古方记载和民间长期食用证实具有良好的消炎止肿作用，但由于自然分布的地域性，国内外对其生物活性物质的研究还较少，因此对荔枝螺药效物质基础和作用机制的探究具有重要意义。 本项目以福建海域荔枝螺为研究材料，通过蜂毒磷脂酶抑制，NF-kB报告基因激活以及p65入核观察等手段指导分离荔枝螺抗炎组分（E-EA-3），该活性组分显著抑制LPS诱导的RAW 264.7 中TNFα 、IL-1β等炎性因子 mRNA水平及蛋白水平的上调，抑制LPS诱导下IκBα的降解和JNK蛋白的磷酸化，细胞从而通过抑制NF-ΚB信号通路有效拮抗LPS所致炎症激活作用。不同剂量荔枝螺提取物在足肿胀大鼠炎症模型上也表现出良好的抗炎活性。另一活性组分E-EA-2证实具有良好的抗癌活性。E-EA-2有效抑制乳腺癌细胞MCF-7，肝癌细胞QGY-7703增殖， 药物浓度高于40 μg/ml 时，两种癌细胞存活率显著降低（<56%）。E-EA-2能诱导肝癌细胞QGY-7703发生PARP蛋白剪切，加入泛Caspase抑制剂使细胞存活率提高了26%。同时，E-EA-2使MCF-7细胞核产生固缩，线粒体膜电位显著下降，凋亡细胞数量显著增加。E-EA-2对肝癌细胞QGY-7703的IC50为46μｇ/ml，对乳腺癌细胞MCF-7的IC50为28μｇ/ml，对癌细胞的增殖抑制作用主要是以促凋亡方式实现的。 通过硅胶柱层析、制备型液相色谱等手段从荔枝螺腺体乙酸乙酯提取物中分离纯化出3个含溴化合物，纯度达98%，通过核磁共振、高分辨质谱等对其进行了结构鉴定。