补肾益精中药治疗肾精亏虚证相关疾病的生物学物质基础及作用机制的预测与验证

Prediction and verification of biological basis and mechanism for traditional Chinese drugs of reinforcing kidney for supplementing essence in treating diseases related to deficiency of kidney essence

"肾精"是中医学的重要物质概念,但其实质至今未明。补肾益精中药治疗肾精亏虚证的临床应用极其广泛,但其生物学物质基础及其作用机制至今未被阐明。本文拟探寻多种补肾益精中药治疗多种肾精亏虚证疾病的共同作用的重要靶点,以期阐明其作用机制,并对阐释"肾精"本质提供参考。本文首先采用网络药理学方法,依据TCMSP、DisGeNET、Uniprot等数据库及研究报道文献资料,构建3味经典补肾益精中药(熟地黄、制首乌、黄精)与5种公认的肾精亏虚证疾病(骨质疏松症、阿尔茨海默症、贫血、不孕不育症、少精症)的"(补肾益精功能)药物-成分-靶点-疾病(肾精亏虚证候)"的关联网络。从该网络中筛选出关键靶点群,利用Clue GO和DAVID数据库对其进行生物功能注释。最后采用自然衰老肾精亏虚证小鼠模型,与青年小鼠、灌胃熟地黄的模型小鼠对比,验证上述网络分析所获得的重要靶点。小鼠的饲养和使用均符合中国《实验动物管理条例》及西南大学药学院实验动物伦理委员会相关规定。网络药理学分析结果发现,"(补肾益精功能)药物-成分-靶点-疾病(肾精亏虚证)"关联网络存在175个中药与疾病的相同靶点,经分析网络拓扑参数获得71个关键靶点。这些靶点涉及转录、RNA代谢、DNA依赖的转录调控等多种生物过程;核腔、细胞器内腔、膜封闭腔等细胞成分;转录调节、转录因子活性、酶结合等分子功能,以及参与过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptor alpha,PPARα)、丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)、缺氧诱导因子1(hypoxia-inducible factor 1,HIF-1)和促红细胞生成素(erythropoietin,EPO)等信号通路调控。动物实验验证结果显示,自然衰老肾精亏虚证模型小鼠EPO含量及其通路中HIF-1α、生长因子受体结合蛋白2(growth factor receptor-bound protein 2,GRB2)、MAPK3、信号传导及转录激活蛋白5A(signal transducer and activator of transcription 5A,STAT5A)、转录因子AP-1(transcription factor AP-1,JUN)和原癌基因c-Fos(proto-oncogene c-Fos,FOS)表达量均显著降低,熟地黄能显著逆转上述各项蛋白的降低。网络药理学预测结果与模型动物验证结果均表明,补肾益精中药治疗肾精亏虚证相关疾病的作用靶点及机制,可能与调节细胞核内转录活性及EPO信号通路有关。

"Kidney essence"is a profound concept in the theory of traditional Chinese medicine.But its biological basis is unknown until now,resulting in the therapeutic effects of traditional Chinese drugs on reinforcing kidney for supplementing essence hard to be evaluated.This study aimed,to explore the potential biological basis and mechanism of traditional Chinese drugs of reinforcing kidney for supplementing essence on diseases related to deficiency of kidney essence through network pharmacology analysis on the intersection of targets of drugs and diseases.The targets for ingredients in Rehmanniae radix praeparata(RRP),Polygoni multiflori radix praeparata(PMRP)and Polygonati rhizome(PR)were gathered from TCMSP and TCMID database.Osteoporosis,Alzheimer’s disease,anemia,infertility and oligospermia targets were collected from OMIM and DisGeNET database.Drugcompound-target-disease(DCTD)network was established with Cytoscape 3.6.1 software,then Clue GO and DAVID database was used to acquire the annotation about GO terms and signaling pathways.Natural aging mice,an acknowledged syndrome model of deficiency of kidney essence,and RRP were used to verify the predictive targets by Western blot analysis.All animal experiments were conducted in accordance with the international guidelines and regulations for the care and use of animals.DCTD network showed that the intersection of drugs and diseases included 175 common targets.After topology analysis,71 key were screened out targets which were associated with GO annotation exhibited that biological processes(including transcription regulation,RNA metabolism regulation,and DNA-dependent transcription regulation),cell composition(including nuclear lumen,organelle lumen,and membrane closure lumen),molecular function(including transcription regulation,transcription factor activity,and enzyme binding),and signaling pathway(including peroxisome proliferator-activated receptor alpha(PPARα),mitogen-activated protein kinase(MAPK),hypoxia-inducible factor 1(HIF-1),erythropoietin(EPO)and other signaling pathways.In natural aging mice,the expressions of HIF-1α,growth factor receptorbound protein 2(GRB2),MAPK3,signal transducer and activator of transcription 5 A(STAT5 A),transcription factor AP-1(JUN)and proto-oncogene c-Fos(FOS)in EPO pathway were significantly decreased.RRP significantly reversed the decrease of the above targets.Above all,these results indicated that the therapeutic effects of traditional Chinese drugs of reinforcing kidney for supplementing essence on deficiency of kidney essence may be related to the regulation of nuclear transcriptional activity and EPO signaling pathway.