基于关键细胞信号转导效应靶标建立抗肝纤维化中药的生物活性评价与质量控制新方法

Establish a New Bioactivity Evaluation and Quality Control Method Based on Key Cell Signal Transduction Target

中药质量是制约中医药发展的重要瓶颈,指纹图谱等基于成分分析的中药质控方法存在效应成分不明及与活性作用不符等局限,亟需发展基于生物活性评价的新方法。立足于扶正化瘀方抗肝纤维化的长期临床与药理研究积累,以及国际临床试验的重要需求,本文提出以下假说:扶正化瘀存在抗肝纤维化的主要药理靶标群,药物对该靶标群基因转录活性的影响,可良好反映其抗肝纤维化药效。首先,本研究选择肝星状细胞、肝内皮细胞、肝细胞与巨噬细胞等4种细胞系,考察溶媒、作用浓度与时间等因素对扶正化瘀细胞效应的影响,建立扶正化瘀体外标准给药方法;其次,基于细胞信号通路,蛋白芯片与网络药理相结合发现扶正化瘀抗肝纤维化的关键药理靶标;最后,构建表征中药靶标基因转录活性的报告基因质粒与稳转细胞株,比较不同批次扶正化瘀对靶标的基因转录活性、细胞生物活性与模型动物肝纤维化的影响,并指纹图谱分析其成分。以建立抗肝纤维化中药的生物活性评价与质量控制新方法,促进中药产品质量提高与创新药物研发。

The quality of traditional Chinese medicine(TCM)is the significant bottleneck for the development of TCM modernization.TCM quality control like fingerprint,which based on chemical analysis,has limits such as the unknown active components and the inconsistence between chemical components and activity.Therefore,new bioactivity evaluation-based quality control method is of great necessary.Based upon the long-term accumulation of clinical experiences and pharmacological mechanism research of Fuzheng Huayu(FZHY)in our research group,as well as the significant demand in international clinical trials,we hereby propose the following scientific hypothesis:FZHY has the major pharmacological targets for anti-hepatic fibrosis.The FZHY influence on transcriptional activity of those pharmacological targets,may reflect its anti-hepatic fibrosis effect well.Firstly,four cell lines,hepatic stellate cell,hepatic endothelial cell,hepatic cell and macrophage are selected to investigate the cell effect influenced by solvent,working concentration and time,so that a standard in-vitro drug delivery system will be optimized and established.Secondly,we try to discover the key pharmacological target of FZHY on liver fibrosis in terms of cell signaling pathway,protein chip and network pharmacology.Finally,recombinant reporter gene plasmid containing target gene promoter and stably transfected cell lines will be established and different batches of FZHY will be evaluated and compared on target gene transcription activity,cell bioactivity and anti-hepatic fibrosis effect in animal model.Meanwhile,components of FZHY will be analyzed as well.The purpose is to establish a new bioactivity evaluation-based quality control method for anti-hepatic fibrosis TCM,to provide new technical platform for the improvement of TCM quality and the discovery of novel drug.