A network pharmacology study of Chinese medicine QiShenYiQi to reveal its underlying multi-compound, multi-target, multi-pathway mode of action

Abstract

Chinese medicine is a complex system guided by traditional Chinese medicine (TCM) theories, which has proven to be especially effective in treating chronic and complex diseases. However, the underlying modes of action (MOA) are not always systematically investigated. Herein, a systematic study was designed to elucidate the multi-compound, multi-target and multi-pathway MOA of a Chinese medicine, QiShenYiQi (QSYQ), on myocardial infarction. QSYQ is composed of Astragalus membranaceus (Huangqi), Salvia miltiorrhiza (Danshen), Panax notoginseng (Sanqi), and Dalbergia odorifera (Jiangxiang). Male Sprague Dawley rat model of myocardial infarction were administered QSYQ intragastrically for 7 days while the control group was not treated. The differentially expressed genes (DEGs) were identified from myocardial infarction rat model treated with QSYQ, followed by constructing a cardiovascular disease (CVD)-related multilevel compound-target-pathway network connecting main compounds to those DEGs supported by literature evidences and the pathways that are functionally enriched in ArrayTrack. 55 potential targets of QSYQ were identified, of which 14 were confirmed in CVD-related literatures with experimental supporting evidences. Furthermore, three sesquiterpene components of QSYQ, Trans-nerolidol, (3S,6S,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol and (3S,6R,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol from Dalbergia odorifera T. Chen, were validated experimentally in this study. Their anti-inflammatory effects and potential targets including extracellular signal-regulated kinase-1/2, peroxisome proliferator-activated receptor-gamma and heme oxygenase-1 were identified. Finally, through a three-level compound-target-pathway network with experimental analysis, our study depicts a complex MOA of QSYQ on myocardial infarction.

Figure 1. Effects of three sesquiterpene compounds from volatile oil of Dalbergia odorifera T. Chen on NO production in LPS-stimulated RAW264.7 macrophages.

(A) Structure of ENL, SDL and RDL isolated from Dalbergia odorifera T. Chen. (B) Effects of ENL, SDL and RDL on RAW264.7 macrophages viability. (C) Effects of ENL, SDL and RDL on NO production in LPS-stimulated RAW264.7. Indo stands for indomethacin and was used as positive control. Each group was compared with model, and * stands for p<0.05, ** stands for p<0.01. Each assay was conducted in triplicate and repeated three times. ENL is trans-nerolidol; RDL is (3S,6R,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol; SDL is (3S, 6S, 7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol.

Figure 2. Effects of three sesquiterpene compounds from volatile oil of Dalbergia odorifera T. Chen on pERK1/2, ERK1/2, PPARγ and HO-1 expression in LPS-stimulated RAW264.7 macrophages.

(A). LPS co-incubation induced significant increase of pERK1/2 expression, the ratio of pERK1/2 to ERK1/2 was higher than that in the control group (*p<0.05). 50 µM ENL attenuated the increased ratio of pERK1/2 to ERK1/2, while 50 µM SDL suppressed this ratio compared to LPS treated alone (#p<0.05), and RDL had no effect on the increased ratio. (B). LPS decreased PPARγ expression (**p<0.01 vs. control), and ENL, RDL, SDL all partially increased PPARγ expression, ENL showed the most strong effect (#p<0.05 vs. model). (C). The expression of HO-1 was induced to increase with 200 ng/ml LPS stimulation (**p<0.01 vs. control), and ENL, RDL, SDL further promoted HO-1 expression (for ENL p<0.05, and for RDL, SDL p<0.01, comparing with model). The RAW264.7 was stimulated with 200 ng/ml LPS for 24 h, and the concentration of ENL, RDL and SDL were all at 50 µM. Each assay was conducted in triplicate and repeated three times. ENL is trans-nerolidol; RDL is (3S,6R,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol. SDL is (3S, 6S, 7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol.

Figure 3. Effects of U0126, T0070907, and Pioglitazone hydrochloride (Pio) on NO production in LPS-stimulated RAW264.7 macrophages.

LPS significantly induced NO secretion (^§§ p<0.01 vs control). ERK1/2 phosphorylation antagonist U0126 significantly inhibited NO production (**p<0.01 vs. LPS treated alone) (A and D). PPARγ phosphorylation inhibitor T0070907 strongly suppressed NO release (**p<0.01 vs. model) (C), while PPARγ agonist Pio decreased NO secretion (**p<0.01 vs. model) (B). ENL does-dependently attenuated NO secretion, U0126, T0070907, and Pio further strengthened ENL's effect. 50 µM SDL also showed NO inhibition activity, while U0126 enhanced SDL's effect. * Means compared with model group (200 ng/ml LPS treated alone), and # stands for statistic significance analysis between two groups with and without small molecular tool drugs (i.e. U0126, T0070907, and Pio). ENL is trans-nerolidol; RDL is (3S,6R,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol.

Figure 4. Multi-compound-multi-target-multi-pathway network of TCM QSYQ on treating myocardial infarction.

There were 52 differentially expressed genes which were validated in published literatures. Node size of targets and pathways are proportional to the number of compounds associated with the node. This network depicted a clear three-level structure of the mode of action of main compounds of QSYQ, that is multi-compound regulated multi-pathways through modulating of groups of genes to show their pharmacological and clinical effects. Compound was linked to targets, while target were linked to pathways, a certain compound linked indirect link to pathways through certain targets. Diamond represents compounds; Circle represents target genes; Vee represents CVD related pathways. The edge went through compound to target, and target to pathway.