Pathway as a pharmacological target for herbal medicines: an investigation from reduning injection

Abstract

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.

Fig 1. Workflow for signaling pathway-based strategy.

Fig 2. Drug-target (D-T) network.

Circles and round rectangles correspond to the target proteins and drugs, respectively. Target nodes are colored according to their protein families. A link represents the interaction between a drug and a target node based on an in-house method (RF value ≥ 0.7 or SVM ≥ 0.8).

Fig 3. Cell viability of RAW 264.7 cells (4×105 cells/ml) in response to RDN and LPS.

The viability of RAW 264.7 cells was determined by CCK-8 assay after incubated with indicated concentrations of RDN for 24 h supplemented with or without 1 μg/ml of LPS. **p < 0.01 represents significant difference compared with the control group.

Fig 4. Effects of RDN on the production of NO, iNOS mRNA and protein in RAW 264.7 cells.

Cells were pre-incubated with or without RDN at various concentrations for 2 h and then stimulated with LPS (1 μg/ml) for an indicated time. NO production (A) in culture medium was determined after cultured with LPS for 24 h, the gene expression of iNOS (B) was measured by quantitative real-time PCR after stimulated with LPS (1 μg/ml) for 8 h, and the protein level of iNOS (C) in whole cell extracts were determined after being stimulated for 18 h by western blot. Data are presented as mean ± standard error of three independent experiments in triplicate. *P < 0.05 and **P < 0.01 represent significant difference when compared with LPS group.

Fig 5. The mRNA expression of pro-inflammatory cytokines in RAW 264.7 cells.

RAW 264.7 cells pretreated with 2-fold serial diluted RDN for 2 h were cultured in absence or presence of LPS (1 μg/ml) for 8 h. mRNA levels of TNF-α (A), IL-1β (B) and IL-6 (C) were quantified by quantitative real-time PCR. Data were presented as mean ± standard error of three independent experiments in triplicate. *P < 0.05 and **P < 0.01 represent significant difference compared with the cells that treated with LPS only.

Fig 6. Effects of RDN on LPS-induced NF-κB activation in RAW 264.7 cells.

Cells were pretreated with different doses of RDN for 2 h, followed by stimulated with LPS (1 μg/ml) for 30 min. The phosphorylated p65, IκB-α and total p65, IκB-α were immunoblotted with specific antibodies, with β-actin being used as control. Results were repeated through three independent experiments with the same tendency.

Fig 7. Effects of RDN on LPS-induced MAPKs activation in RAW 264.7 cells.

Cells were pretreated with control solution or RDN for 2 h, followed by incubation with or without LPS (1 μg/ml) for a fixed time. Phosphorylated ERK1/2, p38 and JUK as well as non-phosphorylated proteins were detected after being incubated for 30 min (A). And after 18 h, the proteins of ERK1/2 and c-Jun regardless of phosphorylated or not were also examined (B). And (C) depicts the putative modulation pathways that respond to RDN therapeutic molecular mechanisms in LPS-stimulated RAW 264.6 cells, where blue downward arrows and red upward arrows represent decreased and increased tendency of a target respectively.

Fig 8. The joint-action effects of C, G and S.

Cells were pre-incubated for 2 h with or without test agents, and then incubated with LPS (1 μg/ml) for an indicated time. The joint-action effect on NO production (A and B) was examined after stimulated with LPS for 24 h, and 18 h for COX-2 and iNOS (C) expression, respectively.