To Unveil the Molecular Mechanisms of Qi and Blood through Systems Biology-Based Investigation into Si-Jun-Zi-Tang and Si-Wu-Tang formulae

www.nature.com/scientificreports OPEN received: 10 June 2016 accepted: 12 September 2016 Published: 28 September 2016 To Unveil the Molecular Mechanisms of Qi and Blood through Systems Biology-Based Investigation into Si-Jun-Zi-Tang and Si-Wu-Tang formulae Jing Sun1,*, Li Zhang2,*, Yujun He3, Kun Zhang3, Liping Wu3, Yongsheng Fan3 & Zhijun Xie3 Traditional Chinese Medicine (TCM) is increasingly getting clinical application worldwide. But its theory like QI-Blood is still abstract. Actually, Qi deficiency and blood deficiency, which were treated by Si-JunZi-Tang (SJZT) and Si-Wu-Tang (SWT) respectively, have characteristic clinical manifestations. Here, we analyzed targets of the ingredients in SJZT and SWT to unveil potential biologic mechanisms between Qi deficiency and blood deficiency through biomedical approaches. First, ingredients in SWT and SJZT were retrieved from TCMID database. The genes targeted by these ingredients were chosen from STITCH. After enrichment analysis by Gene Ontology (GO) and DAVID, enriched GO terms with p-value less than 0.01 were collected and interpreted through DAVID and KEGG. Then a visualized network was constructed with ClueGO. Finally, a total of 243 genes targeted by 195 ingredients of SWT formula and 209 genes targeted by 61 ingredients of SJZT were obtained. Six metabolism pathways and two environmental information processing pathways enriched by targets were correlated with 2 or more herbs in SWT and SJZT formula, respectively. Traditional Chinese Medicine (TCM), being an effective treatment system, is increasingly getting clinical application worldwide. After more than 5000 years of clinical practice, nearly 100,000 classical and effective TCM formulae have been developed, but the effective mechanisms of most formulae remain unclear1. The abstract and subjective theories like Yin-Yang and QI-Blood theories are still the main obstacle for application of TCM worldwide. Actually, Qi deficiency and blood deficiency have characteristic clinical manifestations. The clinical manifestations should be based on objective pathological change at gene or protein level. Similar with western medicine, ingredients of Chinese medicine have their targets (proteins or genes, etc.), which is the key factor to bridge the gap between western medicine and TCM. Since the TCM formulae are normally composed of several medicinal herbs, and each herb normally has many ingredients, and each ingredient has a lot of targets, a formula is a complex biologic active network. Fortunately, along with the rapid development of life science and computer science, a variety of computational tools and bioinformatic database have been developed to facilitate the analysis of a large number of genes associated with complex ingredients of TCM formulae2, which provide opportunities to predict potential pharmacological actions of TCM formulae and clarify complex molecular mechanisms of formulae and theories of TCM. Based on primary biomolecular databases, e.g. Kyoto Encyclopedia of Genes and Genomes ( KEGG, http://www.kegg.jp)3, HPRD4, PDB5, TTD6, OMIM7, Drug-Bank8, STITCH9 and ChEMBL10, a lot of TCM-related databases have been developed, such as TCMID11, HIT12, TCM Database@Taiwan13, TCMGeneDIT14, TCM-ID15, TCMSP16 and CHMIS-C17. These TCM-related databases complement each other to provide information on complex interactions of TCM-active ingredient-gene-disease2. Among these TCM-related databases, TCMID (http://www.megabionet.org/tcmid/) contains 3,791 diseases, 47,000 prescriptions, 8,159 herbs, 6,828 drugs, 25,210 compounds and 1 The Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China. 2Hangzhou Traditional Chinese Medical Hospital, Hangzhou, Zhejiang, China. 3Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.F. (email: ) or Z.X. (email: ) Scientific Reports | 6:34328 | DOI: 10.1038/srep34328 1 www.nature.com/scientificreports/ Figure 1. Research flow chart. We analyzed targets of the ingredients in Si-Jun-Zi-Tang (SJZT) and Si-Wu-Tang (SWT) to unveil the difference and relationship in molecular biological mechanisms between Qi deficiency and blood deficiency through biomedical approaches. First, ingredients in SWT and SJZT were retrieved from TCMID database. Then a visualized network of KEGG pathways was constructed with ClueGO to unveil the difference and relationship between Qi and blood. 17,521 related targets, which facilitates the study of interactions between formula, ingredient, gene and disease to uncover the molecular biological mechanisms of TCM. Meanwhile, there are several network analysis tools for biological functionality of TCM-related network analysis, such as Cytoscape18,19. More than 150 specialized plugins integrated in Cytoscape can be used to import and map existing interaction data cataloged in public databases2, such as ClueGO20, BioGrid Plugin21 and MiMI22. ClueGO integrates Gene Ontology (GO) terms as well as KEGG/BioCarta pathways to create functionally organized GO/pathway term network and analyze one or compare two lists of genes and comprehensively visualizes functionally grouped terms. Increasing TCM researchers successfully analyzed pharmacological mechanism of TCM formulae by using TCM-related databases and data analysis tools23–26. In trying to unveil the different potential biologic mechanisms between Qi deficiency and blood deficiency through biomedical approaches, we chose SJZT and SWT, which have been used in China and other Asian countries for about 1,000 years to effectively rectify Qi deficiency and blood deficiency, respectively. SJZT consisting of Panax ginseng, Atractylodes macrocephala, Poria cocos and Radix Glycyrrhizae Preparata, is the basic TCM prescription of tonifying Qi. SWT consisting of Rehmannia glutinosa, Angelica Sinensis, Ligusticum chuanxiong and Paeonia albiflora, is the TCM classical prescription of nourishing blood. SJZT and SWT were both recorded in < Taiping and the agent of the bureau party >  , which was the first national pharmacopoeia and was published in twelfth Century in the Song Dynasty of China. The research flow chart was shown in Fig. 1. Results Ingredients and targets of SWT and SJZT. As shown in Retrieving from TCMID, we obtained 152 genes targeted by 162 ingredients of Radix Angelicae Sinensis, 107 genes targeted by 28 ingredients of Ligusticum chuanxiong, 21 genes targeted by three ingredients of Rehmannia glutinosa, 42 genes targeted by eight ingredients of Paeonia albiflora (Fig. 2A), and 156 genes targeted by 37 ingredients of Panax ginseng, nine genes targeted by two ingredients of Atractylodes macrocephala, nine genes targeted by one ingredients of Poria cocos and 62 genes targeted by 22 ingredients of Radix Glycyrrhizae Preparata (Fig. 2B). After screened according to STITCH combined-score more than 0.7, a t (...truncated)