金英黄归汤对乳腺上皮细胞胞外LPS-TLR4信号途径的影响

Mammary epithelial cells are involved in the immune defense against infection, Which plays a pivotal role in the occurrence of mastitis and development, induced by invading pathogens. Nowadays, extracellular LPS-TLR4 signaling are rarely reported in mammary epithelial cells, the research of Chinese medicine's effect on it are less. In this project, firstly, we'll screen out the most suitable concentration of LPS binding target cells by Flow cytometry and detect the expression of CD14 and MD-2 in mammary epithelial cells by Laser Scanning Confocal Microscope or PCR, on this basis, study effects of the two co-receptor proteins on LPS binding target cells with the addition of CD14 and MD-2 antagonists or the methods of siRNA transfection. Secondly, examine effects of jin-ying-huang-gui preparation and the active ingredients on the related factor of LPS/TLR4 signaling in mammary epithelial cells, of which , we need to detect the content of TNF-α and IL-8 by ELISA, target cell binding ratio and the mRNA or protein expression of CD14 and MD-2 by Flow cytometry or Fluorescent Quantitative RT-PCR. Finally, detect effects of the drugs on LPS and TLR4-MD-2-LPS complex structure by X-ray diffraction. The above research contributes to further confirm extracellular LPS-TLR4 signal pathway in mammary epithelial cells, reveal the effects of traditional Chinese medicine and the active ingredients on the signal pathway at molecular level so as to solve the problem that the mechanisms of Chinese medicines is unknown . we hope the above research can provide theoretical data for the clinical treatment of dairy cow mastitis.

乳腺上皮细胞对感染做出识别和应答对于乳房炎的发生发展尤其重要，目前细胞胞外LPS-TLR4信号途径的研究报道甚少，中药对其影响的报道也少。本项目拟采用流式细胞术筛选LPS结合细胞的适宜浓度，间接免疫荧光法或PCR技术检测细胞CD14、MD-2表达，通过CD14、MD-2的拮抗剂或通过siRNA转染研究它们对LPS结合细胞的作用，通过流式细胞术检测细胞结合率、ELISA法检测TNF-α、IL-8含量、流式细胞术或实时荧光RT-PCR检测CD14、MD-2蛋白或mRNA表达共同研究金英黄归汤及绿原酸对乳腺上皮细胞LPS-TLR4信号途径相关因子的影响，采用X-单晶衍射检测药物对LPS结构变化的影响。研究结果将明确乳腺上皮细胞胞外LPS至TLR4信号途径，并从分子水平揭示中药对其的作用机制，解决中药作用机制不明确的问题，为临床治疗奶牛乳房炎提供理论数据。

本项目考察了脂多糖（LPS）刺激乳腺上皮细胞（MECs）最佳用量，确定MECs上CD14、MD2的表达及对胞外LPS-TLR4信号途径的作用，考查了金英黄归汤（JYHGP）及其主要成分绿原酸分别对类脂A的结构、LPS与细胞的结合率、LPS介导细胞因子分泌的影响，以及乳腺上皮细胞胞外LPS-TLR4信号途径相关因子的影响。另外，从临床上获得一株猪源大肠杆菌，尝试对其LPS的提取、纯化及活性进行研究，并提取分离纯化、鉴定了这株大肠杆菌的类脂A。.研究发现，LPS作用于小鼠MECs的 IC50为76.83µg/mL，确定5-10 µg/mL LPS作用细胞6h-24h为最佳实验条件，细胞中存在CD14、MD2的基因表达，细胞膜上有大量MD2蛋白表达，而CD14蛋白表达量较少，CD14、MD2基因沉默影响了LPS与细胞的结合率，同时也影响了细胞分泌IL-6、TNF-α及IL-1β等前炎症细胞因子。一定量金英黄归汤、绿原酸能降低LPS与MECs的结合率，能抑制LPS介导细胞分泌TNF-α、IL-6前炎症细胞因子。JYHGP、绿原酸通过抑制小鼠MECs 的胞外CD14、MD2活化来控制细胞因子的释放，这可能是JYHGP、绿原酸的作用机制。另外，从临床上分离获得一株猪源的大肠杆菌，通过培养，提取纯化得到LPS，产率为3.74%，其中蛋白含量为0.48%，多糖含量为13.40%，核酸含量为0.06%，该菌株的LPS含有由不同分子量的结构，说明大分子的LPS的组成及其结构极其复杂，其LPS活性为1.21×107 EU/mg，小鼠LD50为31.86 mg/kg（95%可信区间为26.79～37.89 mg/kg）。以上这些结果明确了MECs胞外LPS至TLR4段的信号途径，特别是CD14、MD-2的作用，并从分子水平揭示中药及有效成分对乳腺上皮LPS至TLR4信号途径各环节的作用机制，为临床上应用中药提供理论数据。