Network Pharmacology-based Study On Mechanism Of Reduning Injection For URTI

Reduning injection is one of TCM injections for treatment of upper respiratory tract infection (URTI) from which is composed of Lonicerae japonicae Flos (Lonicera japonica), Gardeniae Fructus(Gardenia jasminoides), Artemisiae Annuae Herba(Artemisia annua). Clinical studies have revealed a variety of desirable pharmacological effects of Reduning injection on URTI, such as reducing of respiratory viral titres in the airway, suppressing the secretion of inflammatory cytokines (eg PGE2, IL-1, and ET), improving significantly the symptoms of URTI(eg, fever, cough, nasal congestion and sore throat). However, the molecular details about how Reduning injection can be administrated on URTI are still unclear. From the viewpoint of chemical structures, there is a high extent of overlap between Reduning compounds and western drug, which implies Reduning injection are a multi-component, multi-target agents at the molecular level. Therefore, it could be deduced that the therapeutic effectiveness of Reduning injection is achieved through collectively modulating the molecule network of biological systems by its active ingredients. Systems biology, especially network pharmacology, elucidates the underlying mechanisms of biological systems by depicting and studying the complex interactions at different level as various networks, which thus afford possibilities for uncovering the molecular mechanisms related to the therapeutic effect of tradition Chinese medicine. While virtual screening and network analysis provides powerful tools to predict the relationship of compound of TCM and target proteins interactions, to extract hidden information from large-scale data. Applying network pharmacology to the mode of action of Reduning may open up the possibility to understand the interactions the active ingredients in Reduning injection with these targets in the context of molecular networks. Firstly, the database of compound ligand in Reduning injection and target proteins related to URTI is constructed according to the pathogenesis of URTI and the information of the chemical components in Reduning injection. Subsequently, principal component analysis, molecular docking and network analysis were employed to elucidate the action mechanism of Reduning injection, the main active ingredient group and possible indications. According to these predicted results, the whole efficacy of a compound in a biological network is tested to evaluate the activity of its anti-inflammatory and anti-influenza virus molecular. Applying network pharmacology to study the action mechanism of Reduning injection is beneficial not only to our growing understanding of multi-components, multi-targets and multi-pathways mechanism of Reduning injection but also to exploit the molecular mechanism of traditional Chinese medicine.All of the3D structure of the51proteins related to URTI and78chemicals in Reduning injection were obtained from PDB, Bestelin, UNPD database. The chemical molecules has good drug-like and better bioavailability compared with the38drugs for URTI from DrugBank by general purpose module of DiscoveryStudio2.5, and are likely to have some active compounds on URTI by PCA according to the chemical biology principle for similar chemical structure tending to show similar biological activities.After a flexible docking between target proteins and78small molecules was achieved by Autodock4.0, the biological network was constructed by Cytoscape2.8.1including compounds with high docking scores(no less than5and higher than ligand), and then its topological features(network degree, betweenness, network density) were computed by NetworkAnalyzer, a plugin of Cytoscape2.8.1. These results suggest:a) there are25small molecules with a strong interaction with more than3target proteins, of which7chemical molecules with high betweenness target a large number of proteins(>=7) involving in viral replication, secretion of inflammatory mediators and intracellular signal transduction, suggesting that these molecules is of vital importance to Reduning injection for URTI; b) there are four bridging nodes(compounds) in D-D network which occupy critical sites in network and connect subregions to one another, implying that these molecules have the possible synergistic effect in Reduning injection; c) topological analysis of target proteins showed that the molecular mechanism of Reduning injection for URTI symptoms may be involved in inhibiting virus replication (influenza virus, rhinovirus, respiratory syncytial virus, etc.), reducing the secretion of inflammatory cytokines (IL-1, IL-6, NO, PGE2), regulating the MAPKs, PI3K-Akt, JAK-STAT, NF-κB signaling pathway.The inhibition of compounds in Reduning injection on PGE2and NO from RAW264.7by LPS stimulation shows that of the collected28compounds, fifteen compounds significantly suppressed the production of PGE2of which8compounds share the activity of NO, and9compounds reduce cellular concentration of NO. While the predicted results show that, of these active compounds, the anti-inflammatory activities of9compounds might be the results from the suppression of the expression of COX, mPGES-1, iNOS through regulating MAPKs, JAK, NF-κB signaling pathway, and/or the inhibition of PLA2, COX, mPGES-1, iNOS enzyme activity.To further verify the predicted results, Western blot was performed on LPS-induced RAW264.7to investigate the expression of COX-2, mPGES-1, iNOS. The result suggests that the protein expression levels of COX-2, mPGES-1, iNOS are inhibited by four compounds on LPS-induced RAW264.7cells of6compounds involving in regulating the MAPKs, JAK, NF-κB, signaling pathway. Similarly, the anti-influenza activity of compounds in Reduning injection shows that RDN11was confirmed strong antiviral activity against influenza A virus(EC20=28.44μM, SI=24.66) through interacting with multiple stages of the influenza life cycle, such as HA, NA, SAH, IMP dehydrogenase. While network analysis shows that RDN14may interact with HA, IMP dehydrogenase protein and have little influence on the entire biological networks, similarly, RDN14was also confirm the weak inhibition of influenza A virus (EC50=91.284μM, SI=5.48). Basing on these experimental results, it is feasible to investigate the mode action of Reduning injection through the method that molecular docking and analysis is employed to predict the interaction between compounds and target proteins, and the results are tested by pathway-based experiment.