金英黄归汤对小鼠乳腺上皮细胞TLR4信号途径相关因子的影响

It has well established that activation of Toll-like receptors (TLRs) signaling pathways results in the induction of cytokines, which are involved in pro-inflammatory responses and innate immunity. At present, the mechanisms of TLRs signaling pathways have been well characterized in main innate immune cells such as macrophages and dendritic cells. However, which were rarely reported in mammary epithelial cells, the researches of the traditional Chinese medicine's affect to it were less. Our previous studies found that there were TLR2 and TLR4 protein expression in mammary epithelial cells of mouse and lipopolysaccharide (LPS) stimulation of TLR4 transduction could contribute to the release of TNF-α and IL-8,but TLR2 poorly response. To understand this process better, Gene chip technique will be used to search for potential related factors gene expression by mammary epithelial cells in response to exposure to LPS. Meanwhile, in this project we intend to further explore the TLR4 signal pathway mechanism with the methods of siRNA transfection, Western Blot, Immunocytochemistry/ Immunohistochemistry, Gene knockout and ELISA. In addition, active ingredient and content of jin-ying-huang-gui preparation are analyzed by HPLC. And then, we examine the effects of the preparation on the expression of related factors' mRNA in TLR4 signal pathway of mammary epithelial cells, which is stimulated by LPS, by Fluorescent Quantitative RT-PCR. The above research contributes to further confirm TLR4 signal pathway in mammary epithelial cells, broaden the fields of cell function and signal transduction researches, and reveal the effect of the traditional Chinese medicine on the signal pathway at molecular level so as to solve the problem that the targets and mechanisms of the traditional Chinese medicine is unknown. In this project, we hope the above research can provide innovative ideas for the further development of new drug.

TLRs信号转导能促进细胞因子的合成、释放而与前炎症反应和天然免疫相关。目前TLRs信号途径多是对免疫细胞的研究，且仍有不足，乳腺上皮细胞的报道甚少，中药对其影响的报道也较少。我们前期研究发现小鼠乳腺上皮细胞存在TLR2和TLR4，且TLR4转导LPS刺激使细胞分泌TNF-α和IL-8。为此，本项目拟采用基因芯片技术检测LPS刺激下乳腺上皮细胞TLR4信号途径相关因子基因的表达。通过siRNA转染，Western Blot、免疫组化检测,基因敲除，ELISA检测研究TLR4信号的途径。采用HPLC分析金英黄归汤主要活性成分及含量。并采用荧光定量RT-PCR检测中药对信号途径中相关因子mRNA表达的影响。通过研究，明确乳腺上皮细胞TLR4的信号途径，为细胞功能及信号转导等研究拓宽领域，并从分子水平揭示中药对其信号途径的影响机制，解决中药作用靶位和机制不明确的问题，为开发新药提供研究思路。

本项目全面考察了脂多糖（LPS）刺激下的小鼠MECs相关因子在细胞中基因的表达，选择了血小板源性生长因子（Pdgfb）和转化生长因子β（Tgfβ1）通过基因沉默研究其在TLR4信号通路的作用，分析了金英黄归汤（JYHGP）主要有效成分，并建立其质量控制标准，研究了JYHGP及绿原酸、黄芪多糖、连翘苷对小鼠MECs 的TLR4信号途径相关因子的影响。. 研究发现一定量的LPS刺激乳腺上皮细胞后有大量基因差异表达，它们主要参与了代谢、信号转导、转运、炎症应答、免疫应答等生物过程中128个信号通路；其中MAPK信号通路和细胞凋亡通路作用最显著，且与TLR信号通路也关系密切，推断乳腺上皮细胞与免疫细胞TLR4信号途径大体一致。对PDGF-B、TGFβ1基因进行沉默后，其可以显著降低细胞部分细胞炎性因子分泌，它们可能是小鼠MECs上TLR4信号通路中的相关因子。经测定，JYHGP（1g/mL）中总多糖、槲皮素、芦丁、木犀草素、阿魏酸、连翘苷、绿原酸、连翘酯苷A、大黄素的含量平均质量浓度分别为11600 µg/ml、1.69 µg/ml、1.70 µg/ml、0.71 µg/ml、0.95 µg/ml、3.56 µg/ml、3245 µg/ml、3643 µg/ml。提出JYHGP的质控指标为绿原酸、连翘酯苷A和多糖至少分别含有3.10 mg/mL、3.60 mg/mL和10.90 mg/mL。JYHGP通过抑制小鼠MECs 的TLR4/NF-κB信号通路中的TLR4/MyD88/IRAK-1/TRAF-6/TAK-1/NIK途径的活化来控制细胞因子的释放，其也能抑制PDGF-B、Tgfβ1信号途径发挥其作用。活性成分绿原酸、黄芪多糖、连翘苷分别为JYHGP贡献了信号通路中的IRAK-1/TRAF-6/TAK-1/NIK、TLR4/MyD88/IRAK-1/TRAF-6、TLR4/MyD88/TRAF-6/NIK或者TLR4/MyD88/TRAF-6/IκB途径的活化来控制细胞因子的释放，绿原酸也能抑制PDGF-B、Tgfβ1信号途径发挥其作用。以上这些结果明确了乳腺上皮细胞的信号转导途径，并从分子水平全面揭示中药及有效成分对乳腺上皮由TLR4信号途径的作用机制，这将为临床上应用中药提供理论数据。