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.

Introduction

   Chinese medicine, an important branch of the healthcare system in
   China, has gradually gained popularity both at home and abroad [39][1].
   Chinese medicine, a complex system with guidance from traditional
   Chinese medicine (TCM) theories, has proven to be especially effective
   to treat chronic and complex diseases. However, the underlying
   mechanisms of action (MOA) are rarely investigated systematically.

   One of the consensus is that TCM produces its efficacy in a holistic
   way [40][2], taking the advantage of multi-target therapy being more
   effective in combating polygenic diseases than mono-therapies [41][3].
   With the advancements in understanding of pathobiology of human
   diseases, people find that most diseases are not simply caused by one
   single factor [42][4]. It is especially true for the complex
   multifactorial chronic diseases such as cardiovascular disease (CVD),
   cancer and diabetes [43][5], [44][6]. The TCM treatments can be
   visualized as a complexity against complexity paradigm between
   multi-target therapy such as TCM and the complex biological networks of
   human diseases. The development of analytical tools such as systems
   biology [45][7], network biology [46][8] and network pharmacology
   [47][9], [48][10] provide an opportunity to unravel the complex and
   holistic mechanisms of TCM in treating complex diseases. In today's
   post-genomic era, it has become much easier to obtain huge amounts of
   data related to the effect of drugs on transcriptome of target tissues.
   Data analysis becomes critical to identify the MOA of drugs. Recently,
   network models and network analysis of genomic data has been proven
   useful to translate the microarray information and identify potential
   targets [49][11], [50][12] and enriched pathways involved in the MOA
   [51][13]–[52][15]. Tools such as Cytoscape have often been used to
   construct and analyze networks [53][16].

   QiShenYiQi (QSYQ) dropping pills are a Chinese medicine prescription
   approved by the State Food and Drug Administration (SFDA) of China.
   QSYQ is widely and effectively used in China to treat CVD, including
   myocardial infarction, angina, myocarditis, myocardial fibrosis and
   heart failure [54][17]–[55][19]. QSYQ is composed of Astragalus
   membranaceus (Huangqi), Salvia miltiorrhiza (Danshen), Panax
   notoginseng (Sanqi), and Dalbergia odorifera (Jiangxiang). Twelve
   compounds including astragaloside IV (Ast), calycosin (Cal) and
   formononetin (For) from Huangqi; danshensu (DSS), protocatechuic
   aldehyde (PCA) and rosmarinic acid (RSA) from Danshen; ginsenoside Rg1
   (Rg1), ginsenoside Rb1 (Rb1) and notoginsenoside R1 (R1) from Sanqi;
   trans-nerolidol (ENL),
   (3S,6S,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol (SDL) and
   (3S,6R,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol (RDL) from
   Jiangxiang are main components of QSYQ, and they were absorbed into
   blood and distributed into tissues after oral administration [56][20].
   SDL and RDL were identified in Jiangxiang in our previous study
   [57][21]. However, the potential targets and the underlying molecular
   mechanisms of actions of these 12 compounds remain to be systematically
   elucidated.

   In this study, the differentially expressed genes (DEGs) were
   identified from myocardial infarction (MI) rat model treated with QSYQ,
   followed by constructing a CVD related compound-target-pathway network
   linking main compounds to those DEGs supported with literature
   evidences and the pathways that are functionally enriched in
   ArrayTrack. The associations of DEGs with CVD were evaluated based on
   information in database CHD@ZJU of its version 1.0
   ([58]http://tcm.zju.edu.cn/chd/) developed by our group [59][22]. We
   also manually collected the information of targets reported for 9
   compounds (i.e. Ast, Cal, For, DSS, PCA, RSA, Rg1, Rb1 and R1) from
   literatures in PubMed. The judging criteria of a gene being a target is
   that a CVD related DEG can be found directly influenced by a compound
   of QSYQ in literature. Furthermore, as there were no reports on the
   targets of ENL, SDL and RDL, the potential targets of these three
   sesquiterpenes were proposed based upon gene expression data and
   further experimentally validated in this study. Finally, we constructed
   the compound-target-pathway network on anti-MI of QSYQ to decipher the
   underlying multi-compound, multi-target and multi-pathway mechanism.

Materials and Methods

Cell Lines and Reagents

   RAW264.7 cells were purchased from the cell bank of Chinese academy of
   sciences (Shanghai, China). Trans-nerolidol (ENL) was obtained from
   Sigma-Aldrich (Cat No. 18143, Sigma, Germany).
   (3S,6S,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol (SDL) and
   (3S,6R,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol (RDL) was
   extracted from volatile oil of Dalbergia odorifera T. Chen [60][21]
   ([61] Fig. 1A ). Lipopolysaccharide (LPS), U0126 monoethanolate
   (U0126), T0070907, DMSO, and Thiazolyl blue tetrazolium bromide (MTT)
   were obtained from Sigma. Pioglitazone hydrochloride (Pio) was
   purchased from National Institutes for Food and Drug Control (Beijing,
   China). Nitric oxide assay kit was purchased from Beyotime Institute of
   Biotechnology (Cat No. S0021, Haimen, Jiangsu, China). Primary
   antibodies, including anti-ERK1+ERK2 antibody (Cat No. ab17942) for
   ERK1/2, anti-ERK1+ERK2 (phospho T185+T202+Y204+Y187) antibody (Cat No.
   ab4819) for pERK1/2, anti-PPAR gamma antibody (Cat No. ab19481) for
   PPARγ, and anti-Heme Oxygenase 1 antibody [HO-1-1] (Cat No. ab13248)
   for HO-1, were purchased from Abcam (Hongkong, China).

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

   [62]Figure 1
   [63]Open in a new tab

   (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.

Preparation of QiShenYiQi Extracts

   QSYQ was prepared from four herbs, Radix Astragalus membranaceus
   (Chinse name Huangqi), Radix Salvia miltiorrrhiza (Chinse name
   Danshen), Panax notoginseng (Chinse name Sanqi), and Dalbergia
   odorifera T. Chen (Chinse name Jiangxiang). Briefly, crude drug Huangqi
   were crushed, for Huangqi, we added water and extracted with refluxing
   twice, then the extract were filtrated on gauzes. Filtrate was
   concentrated on a rotary evaporator. In the alcohol sedimentation step,
   we slowly added 95% ethanol to the filtrate to make alcohol
   concentration to about 70%. Cooled the solution at room temperature and
   the supernatant was filtered through cotton gauzes. After recycling the
   alcohol, the residue was concentrated again to form Huangqi extract. As
   for crude drug Danshen and Sanqi, they were extracted together with the
   same procedure as that of Huangqi. For Jiangxiang, we boiled crushed
   crude drug Jiangxiang with refluxing, and essential oil was extracted
   in an extraction apparatus. Roots of Sanqi, the trunk and dry heartwood
   of root from Jiangxiang have been used.

Animal Study and Gene Expression Data Analysis

   Male Sprague-Dawley rats (∼250 g) were obtained from Zhejiang
   Experimental Animal Center. The Animal Ethic Review Committees of
   Zhejiang University approved all experimental procedures.

   The rats were maintained in air-conditioned rooms (temperature,
   22–26°C; humidity, 40%–70%) with a 12-h light-dark cycle, and were
   acclimatized to the setting. To provide comfortable conditions for
   rats, each cage contained 5 rats, and the bedding material sawdust was
   renewed every 2 days. Rats were free to get fresh foods and water, and
   the water was renewed everyday. All surgery was performed under 10%
   chloral hydrate anesthesia (300 mg/kg), and all efforts were made to
   minimize suffering. Rat myocardial infarction was made by occlusion of
   left anterior descending coronary artery (LAD) according to Yamaguchi
   [64][23], [65][24]. Rats post-operation were laid flat on an electric
   blanket to help maintain the body temperature of rats. And for those
   rats which were really weak post-operation, we helped them to recover
   heart beating with chest cardiac massage and recover breath by
   connecting the animals with rodent ventilator. For the LAD ligation
   operation, the mortality was about 40%. Six surviving rats after LAD
   ligation were randomly divided into myocardial infarction group (MI)
   and QSYQ-treated group. Three sham-operated rats were treated in the
   same ways as the rats in MI group but without LAD ligation, which were
   served as control group (Control). In clinical, QSYQ pills are
   administered orally administrated to patients with unstable angina and
   heart failure to prevent acute coronary syndrome. In accordance with
   clinical recommendations, QSYQ was given intragastrically (i.g.) at a
   dose of 105.6 mg/kg once a day. Rats in control, and MI groups were
   administered with saline. Rats were sacrificed after 7 days of i.g.
   administration under 10% chloral hydrate anesthesia (300 mg/kg). Rats
   were fixed on a flat plate, and blood samples were collected by
   abdominal aortic method, then the chest was carefully dissected and the
   heart was cut quickly. Three tissue samples on the border between
   infarct and non-infarct area were dissected from left ventricles of
   each group. The tissue samples were stored at −80°C refrigerator.

   Total RNA was extracted using TRIzol Reagent (Invitrogen) and purified
   using RNeasy Mini kit (QIAGEN), following manufacturers' protocols. RNA
   quality was evaluated using an Agilent 2100 Bioanalyzer and
   electrophoresis in 2% (w/v) agarose gels. Only RNA with RNA integrity
   number (RIN) greater than 7.0 and 28S/18S ratio greater than 0.7 was
   used for microarray analyses. Whole genome microarray analysis was
   performed using Affymetrix rat Genome 230 2.0 array. Briefly, total RNA
   were amplified, labeled, and purified using GeneChip 3′IVT Express Kit
   (Affymetrix) according to the manufacturer's instructions to obtain
   biotin labeled cRNA. Array hybridization and wash were performed using
   GeneChip Hybridization, Wash and Stain Kit in Hybridization Oven 645
   (Affymetrix) and Fluidics Station 450 (Affymetrix) following the
   manufacturer's instructions. Slides were scanned by GeneChip Scanner
   3000 and Command Console Software 3.1 with default settings. Spike-in
   control transcripts were monitored to verify hybridization integrity.
   The dataset as CEL files was deposited to GEO and the access number is
   [66]GSE54134. Significant genes were identified through an algorithm
   developed in-house[67][25].

   As mentioned above, we validated the CVD-related DEGs by manually
   checking published literatures in PubMed (by April 10^th, 2013). The
   keywords used to search papers in PubMed were: “Astragaloside IV”,
   “Calycosin”, “Formononetin”, “Danshensu”, “Salvianic Acid A”,
   “Tanshinol”, “Protocatechuic Aldehyde”, “Protocatechualdehyde”,
   “Rosmarinic Acid”, “Ginsenoside Rg1”, “Ginsenoside Rb1”,
   “Notoginsenoside R1”. The literatures related to pharmacology and
   activity of Ast, Cal, For, DSS, PCA, RSA, Rg1, Rb1 and R1 were manually
   read. A DEG was considered as a potential target of a compound if it
   was found to be affected directly by the compound in a paper. For each
   paper, the information of target, PubMed ID, and CVD relevancy were
   recorded. Detailed information on the references used in this study can