Pathway as a Pharmacological Target for Herbal Medicines: An Investigation from Reduning Injection

April Pathway as a Pharmacological Target for Herbal Medicines: An Investigation from Reduning Injection Jianling Liu 0 1 2 Ke Sun 0 1 2 Chunli Zheng 0 1 2 Xuetong Chen 0 1 2 Wenjuan Zhang 0 1 2 Zhengzhong Wang 0 1 2 Piar Ali Shar 0 1 2 Wei Xiao 0 1 2 Yonghua Wang 0 1 2 0 1 College of Life Science, Northwest University , Xi'an, Shaanxi, 710069, China , 2 College of Life Science, Northwest A & F University , Yangling, Shaanxi, 712100, China , 3 Center of Bioinformatics, Northwest A & F University , Yangling, Shaanxi, 712100, China , 4 State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process , Lianyungang, Jiangsu, 222001 , China 1 Data Availability Statement: All relevant data are within the paper 2 Academic Editor: Dermot Cox, Royal College of Surgeons , IRELAND As a rich natural resource for drug discovery, Traditional Chinese Medicine (TCM) plays an important role in complementary and alternative medical systems. TCM shows a daunting complexity of compounds featuring multi-components and multi-targets to cure diseases, which thus always makes it extremely difficult to systematically explain the molecular mechanisms adequately using routine methods. In the present work, to reveal the systematic mechanism of herbal formulae, we developed a pathway-based strategy by combining the pathways integrating, target selection, reverse drug targeting and network analysis together, and then exemplified it by Reduning injection (RDN), a clinically widely used herbal medicine injection, in combating inflammation. The anti-inflammatory effects exerted by the major ingredients of RDN at signaling pathways level were systematically investigated. More importantly, our predicted results were also experimentally validated. Our strategy provides a deep understanding of the pharmacological functions of herbal formulae from molecular to systematic level, which may lead to more successful applications of systems pharmacology for drug discovery and development. - Funding: The research is supported by the Fund of Northwest A & F University (http://www.nwsuaf.edu. cn/) and is financially supported by the National Natural Science Foundation of China (http://www. nsfc.gov.cn/) (Grant No. 31170796 and 81373892). Jiangsu Kanion Pharmaceutical Co. Ltd. provided support in the form of salaries for authors ZW and WX and provided experimental devices and laboratory for this study, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the Traditional Chinese Medicine (TCM) plays an important role in human health through thousands of years of clinical practice [1]. However, the molecular mechanisms involved in TCM still remain somewhat unclear. Herbal medicines are normally designed and featured as multicomponent systems that target multi-targets [2], which makes it hard to decipher the mechanisms of action like those single target drugs by traditional pharmacological methods. Recently, systems pharmacology is increasingly gaining acceptance as a promising way to address the complex problems for herbal medicines [3]. In our previous work, we have successfully built an integrated platform of systems pharmacology and applied it into the discovery of manuscript. The specific roles of these authors are articulated in the author contributions section. bioactive ingredients [4], prediction of drug targets [5], exploration of therapeutic mechanisms [6,7] and reveal of TCM combination rule [8,9], etc. This platform mainly focus on a largescale analysis to find the bioactive compounds and targets and then build drug-disease connections to reveal how herbal medicine actually works on diseases [6]. As we know, the protein targets contained in signaling pathways have been used in drug discovery, and several drugs targeting pathways have reached preclinical and clinical trials for a number of diseases, such as cancer and autoimmune disorders therapy [1012]. But the signaling pathway-interacting networks are rarely highlighted in the existing processes for drug discovery. As a matter of fact, the protein-protein interactions, protein phosphorylation and protein-DNA interactions in signaling pathways often lead to several properties such as extended signal duration, activation of feedback loops and multiple inhibitions [13]. The pathway-based combinatorial therapies which modulate these interactions through a regulation of the target activities in multiple signaling pathways may become a necessity for the maximization of therapeutic efficacy [14]. Thus, the mechanistic studies of signaling pathway networks may offer further therapeutic opportunities for drug discovery and clinical applications. In this work, we provided a systems-based method to explore the therapeutic mechanism of TCM for a given disease based on signaling pathways. As an example, we experimentally verified this method by Reduning injection (RDN) in combating inflammation [15]. This work attempts to offer a novel thinking for deep understanding of biological basis and pharmacology of herbal medicines, as well as a methodology for drug discovery and development. Materials and Methods In slico modeling and analysis The signaling pathways-based approach integrates pathways integration, reverse drug targeting and network construction, and then topological analysis was applied to identify the potential therapeutic mechanism. The detailed descriptions of this tactic are shown in Fig 1. Potential target identification. A large-scale text mining and manual check (from 1995 to 2013) with the keywords inflammation and pathways were performed to acquire the existing pathways that are well identified in the inflammation-associated diseases. Here, NF-B (nuclear factor kappa B) and MAPKs (mitogen-activated protein kinases) pathways are the key players in the progression of inflammation [1618], and the protein IB (inhibitor of kappa B), p65 (transcription factor p65), ERK (extracellular signal-regulated protein kinases), JUN (cJun N-terminal kinases) and p38 (p38 mitogen-activated protein kinases) in these pathways are major or marker drug targets, together with the downstream proteins iNOS (inducible nitric oxide synthase), COX-2 (cyclooxygenase 2) and TNF- (tumor necrosis factor ) [19,20]. The crystal structures of selected targets were fetched from the RCSB Protein Data Bank (http://www.pdb.org/pdb/home/home.do). The PDB IDs of these target proteins are as follows: IKBKB: 4KIK, RELA: 3QXY, MAPK 1: 1TVO, MAPK 3: 2ZOQ, MAPK 8: 4AWI, MAPK 9: 3NPC, MAPK 11: 3GC8, MAPK 12: 1CM8, MAPK 13: 4EYM, MAPK 14: 1OZ1, NOS 2: 3E7G, PTGS 2: 1CVU, TNF: 2AZ5. Reverse drug targeting. Reduning injection is a clinically widely used herbal medicine injection in combating inflammation in China. RDN is composed of three herbs including Artemisiae annuae L. (genus Artemisia, Asteraceae), Gardenia jasminoides J.Ellis (genus Gardenia, Rubiaceae) and Lonicera japonica Thunb. (genus Lonic (...truncated)