基于多靶标膜受体生物色谱微阵列全二维分析系统的抗肝纤维化中药复方活性成分筛选新方法研究

膜受体TβR1、PDGFRβ、TLR4是中药抗肝纤维化的重要靶点。本项目针对当前中药作用膜受体活性成分筛选研究模式灵敏度与专属性的不足和扶正化瘀方抗肝纤维化药效物质不清的问题，在证实该方存在直接作用于肝星状细胞膜TβR1、PDGFRβ、TLR4受体组分的基础上，采用蛋白脂质体重构技术，制备膜受体种类明确、含量可控、构象稳定的生物色谱固定相，建立微阵列TβR1/PDGFRβ/TLR4多靶标集成脂质体全二维色谱活性组分筛选新方法，鉴定扶正化瘀方与三种膜受体的结合组分，评价其抗肝纤维化活性；采用生物色谱竞争实验、表面等离子共振等手段验证结合组分与膜受体相互作用关系，阐明结合组分抗肝纤维化的分子机制。通过本研究，构建多种膜受体高度靶向识别的中药活性成分高通量筛选新方法，不仅有望从中药中同时筛选出具有TβR1/PDGFRβ/TLR4抑制活性的先导化合物，也将为中药活性成分筛选提供新的研究思路与方法。

Membrane receptors TβR1, PDGFRβ, and TLR4 are important targets for traditional Chinese medicine against liver fibrosis. This project focus on the deficiency of the sensitivity and specificity of the current active ingredient screening mode of traditional Chinese medicines, and the unclear pharmacodynamic substances of Fuzheng Huayu Recipe against hepatic fibrosis. Based on the confirmation that there are some components of Fuzheng Huayu recipe directly acting on TβR1, PDGFRβ and TLR4 of hepatic stellate cell, this project will prepare a biochromatographic stationary phase with clear type, controllable content, and stable conformation of membrane receptor by the technique of proteoliposome remodeling, and establish a new method of comprehensive two-dimensional chromatography for screening active components based on microarray of TβR1/PDGFRβ/TLR4 multi-target integrated liposomes, to identify the components of Fuzheng Huayu Recipe binding to three membrane receptors, and then evaluate their anti-fibrotic active. Biochromatography competition experiments, surface plasmon resonance and other means will be used to verify the interaction between the binding components and membrane receptors, and elucidate the molecular mechanism of the binding components against liver fibrosis. Through this study, a new method for high-throughput screening of active constituents of traditional Chinese medicines with highly targeted recognition of multiple membrane receptors was constructed. It is expected to not only screen the leading compounds with inhibitory activity of TβR1/PDGFRβ/TLR4 from traditional Chinese medicine, but also provide new research ideas and methods for the screening of active ingredients of traditional Chinese medicine.