The Synergistic Effects of Astragalus mongholicus and Salvia miltiorrhiza on Coronary Heart Disease Identified by Network Pharmacology and Experiment.

BACKGROUND AND
PURPOSE: Two Chinese herbal medicines Huang Qi (HQ, Astragalus mongholicus) and Dan Shen (DS, Salvia miltiorrhiza) are often combined to treat coronary heart disease (CHD). The purpose of this study was to identify the underlying synergistic effects and mechanisms of HQ and DS against CHD.
METHODS: The active components and targets of HQ and DS, CHD-related genes, and the biological progression were analysed by network pharmacology. The myocardial infarction (MI) rat model was established by ligating the left anterior descending coronary artery. Cardiac function was detected by ultrasonic electrocardiography. The MI size, fibrosis, cardiac hypertrophy, lipid metabolism, blood viscosity, and coagulation indexes were analysed by histological staining or chemical methods, respectively.
RESULTS: A total of 170 shared and specific seed genes of HQ and DS against CHD were identified. The shared and specific biological processes of HQ and DS against CHD were obtained. The LVEF and LVFS values significantly increased, the myocardium infarct size and fibrosis significantly decreased, the values of lipid metabolism indexes and blood viscosity indexes significantly reduced in the HQ + DS treatment group vs HQ or DS single treatment (P < 0.05); the LVEDd, LVEDs, and the CSA values significantly reduced in HQ single and HQ + DS treatment groups vs MI group (P < 0.05); the coagulation index (APTT, PT, TT, and FIB) values decreased significantly in the DS single and HQ + DS treatment groups vs MI group (P < 0.05).
CONCLUSION: In MI rats, HQ and DS exhibited synergistic effects on improving cardiac function, reducing MI size, fibrosis, regulating hyperlipidaemia, and maintaining circulatory system homeostasis; HQ had the specific advantage of alleviating cardiac remodelling; DS had the specific advantage of regulating hypercoagulability. This study revealed that HQ and DS not only exerted synergistic effects but also exhibited complementary effects on CHD.

Introduction

Cardiovascular disease (CVD) remains the leading cause of death and disability worldwide, with increased hospitalisation and discharge rates, causing serious public health issues and economic burden.1 In 2010, the estimated global cost of CVD was US$863 billion, which is expected to increase to US$1044 billion by 2030. Therefore, safe and effective treatments are urgently needed to tackle this major health threat.2

Traditional Chinese medicine (TCM) has attracted significant attention due to its integrative efficacy and fewer side effects, especially on chronic intractable diseases.3 Two Chinese herbal medicines Huang Qi (HQ) and Dan Shen (DS), or specifically Astragalus mongholicus and Salvia miltiorrhiza, respectively, in Latin have been used to treat coronary heart disease (CHD) clinically in China for thousands of years.4,5 According to TCM, patients with CHD often have symptoms of Qi deficiency and blood stasis.6 CHD patients with Qi deficiency often have symptoms of chest tightness, chest pain, palpitation, shortness of breath, fatigue, and pale complexion.7 CHD patients with blood stasis often have symptoms of cyanosis, purple or bruised tongue, fixed chest pain, and sublingual varices.8 Compared with CHD patients without Qi deficiency and blood stasis, larger left ventricular end-diastolic diameter (LVEDd) and left ventricular end-systolic diameter (LVEDs), less left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), and higher blood lipids concentration are present in CHD patients with Qi deficiency and blood stasis.9 Supplementing Qi and promoting blood circulation are the main treatment methods. Invigorating Qi could significantly improve the LVEF, LVEDd, and NTproBNP indexes in chronic heart failure of coronary heart disease.10 Promoting blood circulation treatment can effectively reduce the incidence of angina pectoris and nitroglycerin use, improve heart function, and improve the quality of life of CHD patients.11 HQ has the functions of invigorating Qi, diuresis, detoxification, and regenerating muscles.12 Astragaloside IV (AST), one active component of HQ can protect the heart against MI by improving cardiac histology and ventricular contractility.13 DS has the effects of relieving itching, removing carbuncle, regulating menstruation, stopping bleeding, stabilizing foetus, promoting blood circulation, calming the mind, clearing heat, and avoiding pestilence.14 HQ combined with DS could accelerate blood circulation, prevent thrombosis, eliminate congestion, and remove excess fluid and phlegm. Recently, HQ combined with DS has been applied to treat CVD, type 2 diabetes, pulmonary fibrosis, cirrhosis, and cancer.15–18 Our previous study identified a Chinese herbal formulation Huoxue Anxin recipe (HAR), containing HQ and DS as the monarch herbs, having multi-effects on CHD.19,20 However, the synergistic effects and possible mechanisms of HQ and DS against CHD are not well understood. Identifying the synergistic effects and mechanism of HQ and DS against CHD via modern science and technology would help to clarify these aspects, promote a more extensive clinical application, and benefit individuals who experiencing CHD.

In this study, we identified the specific and shared seed genes of HQ and DS with activity against CHD, and the biological processes of HQ and DS against CHD using network pharmacology. We also verified the synergistic and specific effects and mechanisms of HQ and DS against CHD using experimental evidence (Figure 1).

Figure 1

Process overview. Firstly, the active components of HQ and DS, the shared and specific targets of all the candidate components, the shared and specific seed genes against CHD, and the shared and specific biological processes of HQ and DS against CHD were analyzed by network pharmacology. Secondly, the MI model rats were established by ligating the left anterior descending coronary artery. The synergistic and specific effects of HQ and DS on cardiac function, left ventricular myocardial infarct size, fibrosis, hypertrophy, lipid metabolism, hemorheology, and coagulation were verified by single or combined treating of HQ and DS on MI rats.

Materials and Methods

Network Pharmacology

Active Components Screening of HQ and DS

Chemical components of HQ and DS were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database, a unique pharmacology platform that captures the relationships between herbal ingredients, targets, and diseases (TCMSP, ), the TCM integrated pharmacology database (TCMID, ), and literature mining. The components were filtered by integrating oral bioavailability (OB ≥ 30%) and drug-likeness (DL ≥ 0.18) as suggested by the TCMSP database.21

Target Fishing of the Active Components

Verified targets of the candidate components were retrieved from PubChem () using the criteria of active activity. Predictive targets were retrieved from the SEA database () using the criteria of Max Tc score ≥ 0.5, and from the Swiss Target Prediction database () using the criteria of probability ≥ 0.5.22 In simple terms, the candidate components were uploaded to an online database, the species were restricted to human sources, and potential targets were identified through high-throughput screening. The targets of each component from the different databases were combined and the repeated targets were deleted. The C-T network was visualized using Cytoscape 3.2 software.

The Shared and Specific Seed Genes of HQ and DS Against CHD

CHD-related and differentially expressed (DE) genes were collected using the keywords “coronary heart disease” in the Therapeutic Target Database (), DrugBank (), and DisGeNET () databases which contain collections of genes and variants associated with human diseases.23 The shared and specific seed genes HQ and DS against CHD were obtained by overlapping the shared and specific targets of the HQ and DS with CHD-related DE genes, respectively, using an online Draw Venn Diagram tool ().

Metascape GO Enrichment Based on the Shared and Specific Seed Genes of HQ and DS Against CHD, Respectively

Metascape () enables the automated meta-analysis of gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways. The three lists of genes were retrieved from the latest version of the Metascape database (last updated on 2019-08-14). In the GO Biological Processes (BPs), the highest-level terms in the BP GO graph structure, and pathways with a term ≥3 counts, enrichment factors > 1.5, and a false discovery rate (FDR) < 0.01 were specific biological progress for genes in the network.24 GO analysis was based on the specific and shared seed genes of HQ and DS against CHD, respectively.

Experimental Evidence

Hyperlipidaemia Combined with MI Model in Rats

Male Sprague-Dawley rats (150, bodyweight 180 ± 20 g) were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Animal license: SCXK [Beijing] 2012–0001). The animals were maintained under 12-h light/12-h dark cycles, at a temperature of 22 ± 2°C, and humidity of 50 ± 2%. MI was induced by ligation of the left anterior descending artery, after being given a high-fat forage (cholesterol 1%, egg-yolk powder 10%, lard oil 10%, bile salts 0.2%, and the basic ingredients 78.8%) for 28 days. The protocol was followed as described in our previous report.19,20 Briefly, after the administration of anaesthetics by intraperitoneal injections of 40 mg/kg sodium pentobarbital (Lot. No. 200-323-9, Shanghai Sinopharm Chemical Reagent Co., Ltd, China), the animals were preoperatively subjected to 12-lead electrocardiography (ECG). For the surgical procedure, the rats were subjected to endotracheal intubation with the ventilator set to provide positive pressure ventilation (SAR-830/A, CWE Inc., USA). Following disinfection and opening of the chest, the left anterior descending coronary artery was ligatured 2-mm below the branch point. MI was confirmed with S-T segment elevation of the ECG. The rats in the sham group were similarly operated without the ligation. The 90 surviving rats were randomly assigned into six groups (n = 15): Sham, MI (model), Atorvastatin, HQ, DS, and HQ combined with DS treatment groups. Animals were continuously fed with a high-fat forage diet except for the Sham group. All procedures were carried out following the recommendations of Laboratory Animal Management and Use Regulations from the Beijing Laboratory Animal Management Committee. The protocol was also approved by the Animal Ethics Committee of Guang’anmen hospital, China Academy of Chinese Medical Sciences (approval number: EC_AF_055).

Medical Intervention

Atorvastatin calcium tablets were provided by Pfizer Pharmaceuticals Limited (Lot. No. EG 6081). The tablets were dissolved in distilled water and intragastrically administered at a dose of 7.3 mg/kg/day. The herbal medicines of HQ (Lot. No. 20082311) and DS (Lot. No. 20091081) were provided by Sichuan New Green Pharmaceutical Science and Technology Development Co., Ltd. The granules were dissolved in distilled water. HQ single treatment was administered 5 g/kg/day, DS single treatment was administered 5 g/kg/day, and HQ combined DS treatment was administered with HQ 2.5 g/kg/day + DS 2.5 g/kg/day. The rats were treated intragastrically once a day for four weeks. An equal volume of distilled water was intragastrically administered to the sham and MI groups.

Non-Invasive Transthoracic Echocardiography Measurement

After four weeks of treatment, a non-invasive trans thoracic echocardiography method was used to evaluate cardiac function using a small animal ultrasonic machine (VEVO 3100, VisualSonics, Canada). This method consisted of a two-dimensional mode, a time-motion mode, and blood-flow measurement in the pulsed Doppler mode. The LVEF, LVFS, LVEDd, and LVEDs were calculated from the M-mode.

Anaesthesia, Blood Sample, and Heart Sample Collection

After four weeks of treatment, the rats were anaesthetised by intraperitoneal injections of 40 mg/kg sodium pentobarbital (Lot. No. 200-323-9, Shanghai Sinopharm Chemical Reagent Co., Ltd, China). The abdominal cavity was opened and 9 mL blood samples were collected from the abdominal venous (3 mL for the blood lipid measurements, 4 mL for the blood viscosity measurements, and 2 mL for the blood coagulation measurements). Next, the thoracic cavity was opened and the whole hearts were isolated. In each group, three hearts were quantitatively detected for myocardial infarct size by triphenyltetrazolium chloride (TTC) staining (Lot. No. 20200516, Shanghai Zeye Biotechnology Co., Ltd, China), and five hearts were fixed in 4% paraformaldehyde (Lot. No. 20200713, Solarbio, Beijing, China) for histological analysis.

Determination of Myocardial Infarct Size

Myocardial infarct size was quantitatively detected by TTC staining.25 The hearts were rapidly frozen (−20 °C for 1 h), cut into 4-mm thick slices (total of three), and each slice was incubated at 37°C in 1 mL of 1% TTC (dissolved in pH 7.4 phosphate buffer) for 10 min. The TTC entered the normal cells and bound to dehydrogenase in red, while dead cells were not stained by TTC due to the lack of dehydrogenase. Slices were then transferred to 4% paraformaldehyde for 10 min. Finally, digital photos of the heart slices were obtained (Canon, SX60 HS). The differently stained areas of the heart images (white: infarct region; red: non-infarct region) were quantified by Image-Pro Plus 6.0 (Media Cybernetics, USA). The myocardial infarct size was quantified by measuring the white myocardial area/total white and red myocardial area of the left ventricular×100%. An evaluation of all images was carried out in a blinded manner throughout the study.

Histological Analysis

The rat hearts were fixed for 24 h in 4% paraformaldehyde (Lot. No. 20200713, Solarbio, Beijing, China) at room temperature, embedded in paraffin, and sectioned into 3-μm thick slices from the portion approximately 400-μm distal to the ligation point. Heart sections were deparaffinized using xylene at room temperature and rehydrated in serial dilutions of alcohol. Samples were stained with Masson’s trichrome for the detection of myocardium fibrosis, and wheat germ agglutinin (WGA) for the detection of myocardial cell size.26 Peroxidase conjugated WGA (VECTOR, Catalogue No. PL-1026, USA) 40 mg/mL was diluted in 10 mM phosphate buffer (pH 7.5) and 0.15 M NaCl to produce a working solution of 5 μg/mL. The slides were incubated for 1 hour, then coloured with DAB (Zhongshan Company, Beijing, China) for 10 sec. Each specimen was examined three to four times, and photomicrographs were obtained under a bright field microscope (Olympus IX 70, Japan). The myocardium fibrosis was quantified by measuring the blue myocardium area/total red and blue myocardium area×100%. For the WGA staining, suitable cross-sections, with nearly circular to oval cardiomyocytes, were selected. The outlines of the cardiomyocytes were traced to determine the cross-sectional area (CSA) which was calculated by measuring 400–600 cells distant from the infarct region of each heart.27 The percentage of myocardium fibrosis, and myocardial cell size were observed and imaged under a microscope (Olympus IX 73, Japan), and measured using Image-Pro Plus 6.0 software (Media Cybernetics, USA).

Blood Lipid Measurements

The serum was used to analyse blood lipid indexes. The concentrations of triglycerides (TG, A110-1), total cholesterol (Chol, A111-1), low-density lipoprotein cholesterol (LDL-C, A113-1), and high-density lipoprotein-cholesterol (HDL-C, A112-1) were measured using assay kits from Nanjing Jiancheng Bioengineering (Nanjing, China) according to the kit manufacturer’s instructions.

Blood Viscosity and Coagulation Measurements

A venous blood sample (approximately 4 mL) was collected and mixed well in a 5 mL of heparin vacuum anticoagulation tube. Whole blood viscosity (WBC) and plasma viscosity (PV) at high shear rates (200 /s) were measured using a rotational viscometer (LVDV-II+P, CP40, Brookfield Engineering Labs Inc., USA). Haematocrit (HCT) was measured by a gravimetric technique by centrifuging blood samples at 650 ×g for 20 min. The ratio of HCT to whole blood viscosity (OTE) at high shear rates (200/s) was used as a measure of oxygen availability for tissues.28

A venous blood sample (approximately 2 mL) was collected and placed in a 5 mL vacuum anticoagulant tube containing sodium citrate. The mixture was then centrifuged at 1608 ×g for 10 minutes to separate plasma. Activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB) content were measured using an LG-PABER-I coagulometer (Beijing Steellex Scientific Instrument Company, China) using an automatic coagulation analyser (ACL-TOP700; Instrumentation Laboratory, Milan, Italy) within 24 hours at room temperature. During the measurement, matched calibration products, quality control products, and reagents were used following the manufacturer’s instructions.

Statistical Analysis

Data are presented as the mean ± SD. Statistical comparison of the data was performed using one-way analysis of variance followed by Dunnett’s post hoc test for multiple comparisons using GraphPad Prism 6.0 (GraphPad, Inc., La Jolla, CA, USA). A P value < 0.05 was considered significant.

Results

The Active Components of HQ and DS

A total of 20 active components from HQ and 62 active components from DS were obtained (Table 1). All satisfied the criteria of OB ≧ 30% and DL ≧ 0.18.

Table 1

The Active Components of HQ and DS

HQ
Mol ID	Molecule_name	OB (%)	DL
M438	(3R)-3-(2-hydroxy-3,4-dimethoxyphenyl)chroman-7-ol	67.67	0.26
M33	17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol	36.23	0.78
M380	(6aR,11aR)-9,10-dimethoxy-6a,11a-dihydro-6H-benzofurano[3,2-c]chromen-3-ol	64.26	0.42
M442	1,7-Dihydroxy-3,9-dimethoxy pterocarpene	39.05	0.48
M371	3,9-di-O-methylnissolin	53.74	0.48
M374	5ʹ-hydroxyiso-muronulatol-2ʹ,5ʹ-di-O-glucoside	41.72	0.69
M378	7-O-methylisomucronulatol	74.69	0.30
M379	9,10-dimethoxypterocarpan-3-O-β-D-glucoside	36.74	0.92
M387	Bifendate	31.10	0.67
M417	Calycosin	47.75	0.24
M433	FA	68.96	0.71
M392	Formononetin	69.67	0.21
M296	Hederagenin	36.91	0.75
M398	Isoflavanone	109.99	0.30
M439	Isomucronulatol-7,2ʹ-di-O-glucosiole	49.28	0.62
M354	Isorhamnetin	49.60	0.31
M239	Jaranol	50.83	0.29
M422	Kaempferol	41.88	0.24
M211	Mairin	55.38	0.78
M98	Quercetin	46.43	0.28
DS
Mol ID	Molecule_name	OB	DL
M7132	(3,4-dihydroxyphenyl)acryloyl]oxy-propionic acid	109.38	0.35
M7155	1,6-dimethyl-8,9-dihydro-7H-naphtho[8,7-g]benzofuran-10,11-dione	65.26	0.45
M7150	(6S)-6-hydroxy-1-methyl-6-methylol-8,9-dihydro-7H-naphtho[8,7-g]benzofuran-10,11-quinone	75.39	0.46
M7070	(6S,7R)-6,7-dihydroxy-1,6-dimethyl-8,9-dihydro-7H-naphtho[8,7-g]benzofuran-10,11-dione	41.31	0.45
M7048	(E)-3-[2-(3,4-dihydroxyphenyl)-7-hydroxy-benzofuran-4-yl]acrylic acid	48.24	0.31
M7140	(Z)-3-[2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3,4-dihydroxy-phenyl]acrylic acid	88.54	0.26
M1601	1,2,5,6-tetrahydrotanshinone	38.75	0.36
M7127	1-methyl-8,9-dihydro-7H-naphtho[5,6-g]benzofuran-6,10,11-trione	34.72	0.37
M7050	2-(4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl)-7-methoxy-3-benzofurancarboxaldehyde	62.78	0.40
M7041	2-isopropyl-8-methylphenanthrene-3,4-dione	40.86	0.23
M7059	3-beta-Hydroxymethyllenetanshiquinone	32.16	0.41
M7045	3α-hydroxytanshinone II a	44.93	0.44
M7049	4-methylenemiltirone	34.35	0.23
M2776	Baicalin	40.12	0.75
M7107	C09092	36.07	0.25
M7088	Cryptotanshinone	52.34	0.40
M7082	Danshenol A	56.97	0.52
M81	Danshenol B	57.95	0.56
M7094	Danshenspiroketallactone	50.43	0.31
M7093	Dan-shexinkum d	38.88	0.55
M2651	Dehydrotanshinone II A	43.76	0.40
M7098	Deoxyneocryptotanshinone	49.40	0.29
M569	Digallate	61.85	0.26
M7100	Dihydrotanshinlactone	38.68	0.32
M7101	DihydrotanshinoneI	45.04	0.36
M7105	Epidanshenspiroketallactone	68.27	0.31
M7058	Formyltanshinone	73.44	0.42
M7108	Isocryptotanshi-none	54.98	0.39
M1942	Isoimperatorin	45.46	0.23
M7111	Isotanshinone II	49.92	0.40
M6	Luteolin	36.16	0.25
M7115	Manool	45.04	0.20
M7061	Methylenetanshinquinone	37.07	0.36
M7118	Microstegiol	39.61	0.28
M7119	Miltionone I	49.68	0.32
M7120	Miltionone II	71.03	0.44
M7121	Miltipolone	36.56	0.37
M7122	Miltirone	38.76	0.25
M7123	Miltirone II	44.95	0.24
M7125	Neocryptotanshinone	52.49	0.32
M7124	Neocryptotanshinone ii	39.46	0.23
M7149	NSC 122421	34.49	0.28
M1771	Poriferast-5-en-3beta-ol	36.91	0.75
M1659	Poriferasterol	43.83	0.76
M7130	Prolithospermic acid	64.37	0.31
M7063	Przewalskin a	37.11	0.65
M7064	Przewalskin b	110.32	0.44
M7068	Przewaquinone B	62.24	0.41
M7069	Przewaquinone c	55.74	0.40
M7152	Przewaquinone E	42.85	0.45
M7071	Przewaquinone f	40.31	0.46
M7141	Salvianolic acid g	45.56	0.61
M7142	Salvianolic acid j	43.38	0.72
M7085	Salvilenone	30.38	0.38
M7143	Salvilenone I	32.43	0.23
M7145	Salviolone	31.72	0.24
M7077	Sclareol	43.67	0.21
M2222	Sugiol	36.11	0.28
M7079	Tanshinaldehyde	52.47	0.45
M7151	Tanshindiol B	42.67	0.45
M7156	Tanshinone VI	45.64	0.30
M6824	α-amyrin	39.51	0.76

Notes: The Mol ID, Molecule_name, OB (%), and DL of the active components of HQ and DS were listed.

Abbreviations: OB, oral bioavailability; DL, drug-like.

The Shared and Specific Targets of the Active Components of HQ and DS

A total of 408 targets from 14 active components of HQ and 423 targets from 40 active components of DS were obtained, whereas 6 active components of HQ and 22 active components of DS were no targets reached the set criteria. Among the selective targets, HQ and DS shared 254 targets, HQ had 154 specific targets, and DS had 169 specific targets. The Venn diagram of the shared and specific targets of HQ and DS is exhibited in Supplemental materials (). The C-T network was visualized by Cytoscape software (Figure 2).

Figure 2

C-T network. The C-T network has 629 nodes and 1822 edges. The yellow node in the middle represents the 254 targets shared by HQ and DS. The blue diamond node on the left side represents the specific targets of HQ, and the blue diamond node on the right side represents the specific targets of DS. The pink rectangle node on the left side represents the active components of HQ, the pink rectangle node on the right side represents the active components of DS. Some names of the active components are too long, and represent by molecular ID, eg M33, 17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol; M371, 3,9-di-O-methylnissolin; M378, 7-O-methylisomucronulatol; M442, 1,7-Dihydroxy-3,9-dimethoxy pterocarpene; M1771, poriferast-5-en-3beta-ol; M1601, 1,2,5,6-tetrahydrotanshinone; M7041, 2-isopropyl-8-methylphenanthrene-3,4-dione; and M7049, 4-methylenemiltirone.

The Specific and Shared Seed Genes of HQ and DS Against CHD

A total of 1650 CHD-related DE of genes were collected (1574 from DisGeNET, 68 from Drugbank, and 8 from TTD database). By overlapping the shared and specific targets of HQ and DS with CHD-related DE of genes, HQ and DS shared 83 seed genes against CHD. HQ had 52 specific seed genes against CHD, and DS had 35 specific seed genes against CHD (Figure 3). The UniProt ID, gene name, and protein name of the shared and specific seed genes are listed in the Supplemental materials ().

Figure 3

Venn diagram of the shared and specific seed genes against CHD. The diagram is composed of four sub-sets: HQ T (specific targets of HQ); DS T (specific targets of DS); HQ and DS T (shared targets of HQ and DS); CHD (CHD-related differential genes). The shared seed genes of HQ and DS against CHD are 83. The specific seed genes of HQ against CHD are 52. The specific seed genes of DS against CHD are 35.

Metascape Gene Ontology (GO) Enrichment Analysis Based on the Shared and Specific Seed Genes of HQ and DS Against CHD

The shared biological process of HQ and DS focused on four areas: (1) nutrient metabolism: response to nutrient levels, response to an inorganic substance, metabolism of lipids, lipid biosynthetic process, steroid metabolic process; (2) cell proliferation: genes involved in male infertility, cellular response to organic cyclic compounds, positive regulation of cell migration, positive regulation of ion transport, cellular response to nitrogen, compound Phase I - functionalization of compounds; (3) circulatory system homeostasis: fluid shear stress and atherosclerosis, regulation of hormone levels, circulatory system process; and (4) inflammatory response and oxidative stress: interleukin-4 and interleukin-13 signalling, response to oxidative stress, reactive oxygen species biosynthetic process (Figure 4A). The specific biological process of HQ focused on three areas: (1) nutrient metabolism: response to nutrient levels, vitamin B12 metabolism, small molecule biosynthetic process, AGE-RAGE signalling pathway in diabetic complications, alcohol metabolic process, cellular response to organic cyclic compound, response to purine-containing compound, signalling by receptor tyrosine kinases, Netrin-UNC5B signalling pathway, steroid metabolic process; (2) cell proliferation and fibrosis: positive regulation of cell migration, cellular detoxification, lung fibrosis; (3) inflammatory response and oxidative stress: leukocyte migration, positive regulation of cytokine production, response to reactive oxygen species, reactive oxygen species biosynthetic process (Figure 4B). The specific biological process of DS focused on three areas: (1) lipid and glucose metabolism: lipid catabolic process, cellular response to lipid, response to lipopolysaccharide, glucose homeostasis, cellular response to lipoprotein particle stimulus, monocarboxylic acid transport and metabolic process; (2) circulation system homeostasis: circulatory system process; wound healing; Eicosanoid metabolism via Cyclooxygenases (COX); (3) mood stability: regulation of behaviour, endocrine process (Figure 4C). The detailed information (GO ID, biological process description, gene count, and Log10 P-value) of the GO enrichment is reported in the Supplemental materials ().

Figure 4

GO analysis of the shared and specific biological processes of HQ and DS against CHD. (A), The shared biological processes or pathways of HQ and DS against CHD; (B), The specific biological processes or pathways of HQ against CHD; (C), The specific biological processes or pathways of DS against CHD. The values of the x-axis represent -log 10 P values. The higher the value the more significant the biological processes or pathways.

The Synergistic Effects of HQ and DS on Improving Cardiac Function in MI Rats

The LVEF and LVFS values decreased significantly in the MI group, whereas the LVEDd and LVEDs values increased significantly compared to the values of the sham group (P < 0.01). The LVEF and LVFS values increased in the four treatment groups (Atorvastatin, HQ single, DS single, and HQ + DS), whereas the LVEDd and LVEDs values decreased in two treatment groups (HQ single and HQ + DS) compared to those of the MI group (P < 0.05). Compared to the HQ or DS single treatment group, the values of LVEF and LVFS further increased (P < 0.05) in the HQ + DS treatment group (Table 2 and Figure 5).

Table 2

Effect of HQ and DS on Cardiac Functional Parameters

Groups	n	LVEF (%)	LVFS (%)	LVEDd (mm)	LVEDs (mm)
Sham	15	90.53 ± 9.14	49.90 ± 7.98	6.42 ± 0.41	4.01 ±0.30
MI	11	41.31 ± 6.82**	17.49 ± 4.27**	8.43 ± 0.62**	6.63 ± 0.65**
Atorvastatin	9	51.93 ± 9.75Δ	22.33 ± 4.69Δ	7.95 ± 0.74	5.82 ± 0.71
HQ	12	59.66 ± 7.42Δ	24.61 ± 6.31Δ	7.68 ± 0.57Δ	5.38 ± 0.43Δ
DS	12	56.41 ± 5.31Δ	22.98 ± 6.44Δ	8.05 ± 0.94	5.79 ± 0.86
HQ + DS	13	67.52 ± 7.89ΔΔ#$	32.81 ± 6.12ΔΔ#$	7.62± 0.72Δ	5.24 ± 0.63Δ

Notes: Values are presented as mean ± SD. **P < 0.01 vs the sham group; ΔP < 0.05, ΔΔP < 0.01 vs MI group; #P < 0.05 vs HQ single treatment group; $P < 0.05 vs DS single treatment group.

Abbreviations: LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVEDd, left ventricular end-diastolic diameter; LVEDs, left ventricular end-systolic diameter.

Figure 5

The noninvasive trans-thoracic echo images of different groups The echo images were taken by VEVO 3100, the most advanced equipment for small animal cardiac function detecting: (A), Sham group; (B), MI group; (C), Atorvastatin group; (D), HQ group; (E), DS group; and (F), HQ + DS group. Measurements of the left ventricle dimensions by M-mode tracing of the left ventricle are used for the calculation of LVEF, LVFS, LVEDd, and LVEDs.

The Synergistic Effects of HQ and DS on Reducing Left Ventricular Myocardial Infarct Size

Compared with the sham group, the percentage of infarct size increased significantly (49.51 ± 8.30 vs 0.00 ± 0.00, P < 0.001) in the left ventricle in the MI group. Compared with the MI group, the percentage of infarct size decreased significantly in the left ventricle in four treatment groups (Atorvastatin, HQ single, DS single, and HQ + DS, P < 0.05 or P < 0.01). Compared with the DS single treatment group, the percentage of infarct size decreased significantly in the HQ + DS treatment group (P < 0.05, Figure 6).

Figure 6

The TTC staining and the myocardium infarct size comparison. The photographs of TTC staining: (A), Sham group; (B), MI group; (C), Atorvastatin group; (D), HQ group; (E), DS group; and (F), HQ + DS group. (G), A bar chart of the percentage of the myocardium infarct size among groups. Values are presented as mean ± SD, n = 3. The infarct tissue was indicated in white and healthy cardiac tissue was indicated in red. The myocardial infarct size was quantified by measuring the white myocardial area/total white and red myocardial area of the left ventricular × 100%. ***p <0.001 vs Sham group; Δp <0.05, ΔΔp < 0.01 vs MI group; $P < 0.05 vs DS single treatment group.

The Synergistic Effects of HQ and DS on Alleviating Myocardium Fibrosis and Cardiac Hypertrophy in MI Rats

Compared with the sham group, the number of myocardial cells decreased and collagen accumulation increased significantly (64.26 ± 12.5 vs 2.06 ± 0.86, P < 0.001) in the left ventricular myocardium area in the MI group. Compared with the MI group, the number of myocardial cells increased and collagen decreased significantly in the myocardium area in four treatment groups (Atorvastatin, HQ single, DS single, and HQ + DS, P < 0.05 or P < 0.01). Compared with the sham group, the CSA of left ventricular myocardial cells increased significantly in the MI group (569.33 ± 109.05 vs 280.37 ± 44.06, P < 0.001). Compared with the MI group, the myocardial cell size decreased significantly in two treatment groups (HQ single and HQ + DS, P < 0.05). Compared with the DS single treatment group, the collagen accumulation reduced and CSA of left ventricular myocardial cell size decreased significantly in the HQ + DS treatment group (P < 0.05, Figure 7).

Figure 7

The images of the histological study. (A–F), The photomicrographs of myocardium fibrosis staining with Masson: (A), Sham group; (B), MI group; (C), Atorvastatin group; (D), HQ group; (E), DS group; and (F), HQ + DS group. The fibrotic tissue was indicated in blue and healthy cardiac tissue was indicated in pink. Scale bar = 40 μm. (G–L), The photomicrographs of myocardial hypertrophy staining with WGA (wheat germ agglutinin): (G), Sham group; (H), MI group; (I), Atorvastatin group; (J), HQ group; (K), DS group; and (L), HQ + DS group. The cell membranes were yellow and nuclei were blue. Scale bar = 20 μm. (M), Quantitative analysis of the percentage of myocardium fibrosis; (N), Quantitative analysis of mean cross-sectional area (CSA μm2) of LV cardiomyocytes. Values are presented as mean ± SD, n = 5. ***p<0.001 vs Sham group; Δp<0.05, ΔΔp < 0.01 vs MI group; $P < 0.05 vs DS single treatment group.

The Synergistic Effects of HQ and DS on Improving the Survival Rate

During the experiment, none of the rats died in the sham group; four rats in the MI group died on days 1, 2, 9 and 14 after surgery; six rats in the Atorvastatin group died on days 1, 4, 4, 8, 9, and 12 after surgery; three rats in HQ group died on days 15, 19, and 23 after surgery; three rats in DS group died on days 12, 16, and 21 after surgery; two rats in the HQ + DS group died on days 15 and 18 after surgery. HQ combined with DS treatment significantly increased the survival rate of rats when compared to that of the MI or Atorvastatin group (P < 0.05), but was not considered statistically significant when compared with HQ or DS single treatments (P > 0.05, Figure 8).

Figure 8

The survival rate of rats in the various treatment groups. The survival rate of the different treatment groups was analyzed by the Kaplan–Meier method using GraphPad Prism 6.0 software. The values of the x-axis represent time (0–28 days). The values of the y-axis represent percent survival (0–100%).

The Synergistic Effects of HQ and DS on Lipid Metabolism in MI Rats

Compared with the sham group, the serum TG, Chol, and LDL-C concentrations increased significantly in the MI group (P < 0.01). Compared with the MI group, the serum TG, Chol, and LDL-C concentrations decreased significantly in four treatment groups (Atorvastatin, HQ single, DS single, and HQ + DS, P < 0.05 or P < 0.01), Compared with the HQ single or DS single treatment group, the serum TG, Chol, and LDL-C concentrations further decreased in the Atorvastatin and the HQ + DS treatment groups (P < 0.05, Table 3).

Table 3

The Serum Lipid Indexes Among Groups

Groups	n	TG (mg/dl)	Chol (mg/dl)	HDL-C (mg/dl)	LDL-C (mg/dl)
Sham	15	56.53 ± 10.6	121.75 ± 37.4	64.47 ± 20.42	72.35 ± 26.11
MI	11	185.53 ± 36.17**	239.38 ± 52.34**	48.36 ± 16.52	148.17 ± 38.42**
Atorvastatin	9	68.12 ± 14.20ΔΔ#$	129.65 ± 18.57ΔΔ#$	59.91± 17.33	79.54± 19.25ΔΔ#$
HQ	12	139.45 ± 37.2Δ	148.24 ± 46.75Δ	57.62 ± 12.67	115.16 ± 24.05Δ
DS	12	133.58 ± 25.19Δ	145.22 ± 26.18Δ	58.53 ± 19.35	118.71 ± 21.62Δ
HQ + DS	13	92.41 ± 19.36ΔΔ#$	134.28 ± 23.61ΔΔ#$	57.23± 15.51	83.19 ± 26.74ΔΔ#$

Notes: Values are presented as mean ± SD. **p < 0.01 vs sham group; Δp < 0.05, ΔΔp < 0.01 vs MI group; #P < 0.05 vs HQ single treatment group; $P < 0.05 vs DS single treatment group.

Abbreviations: TG, triglyceride; Chol, cholesterol; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol.

The Synergistic Effects of HQ and DS on Blood Viscosity and Coagulation Indexes in MI Rats

The results of blood viscosity indexes (WBV, PV, HCT, and OTE) and the coagulation indexes (APTT, PT, TT, and FIB) are shown in Table 4. Compared with the sham group, the values of WBV, PV, and OTE were significantly increased (P < 0.01); the values of APTT, PT, and TT were significantly shortened (P < 0.01), and the FIB value was significantly increased (P < 0.01) in the MI group. Compared with the MI group, the values of WBV, PV, and OTE were significantly decreased in four treatment groups (Atorvastatin, HQ single, DS single, and HQ + DS, P < 0.05 or P < 0.01); the values of APTT, PT, and TT were significantly prolonged, and FIB value was significantly decreased in two treatment groups (DS single and DS + HQ, P < 0.05). Compared with the HQ or DS single treatment group, the values of PV were significantly decreased in the Atorvastatin and HQ + DS treatment groups (P < 0.05); the values of WBV and OTE were significantly decreased in the HQ + DS treatment group (P < 0.05).

Table 4

Blood Viscosity and Coagulation Index Among Groups

Groups	n	WBV (mpa/s)	PV (mpa/s)	HCT (%)	OTE (Ratio)
Sham	15	4.37 ± 0.35	1.02 ± 0.47	46.36 ±10.31	0.094 ± 0.007
MI	11	9.57 ± 1.52**	2.19 ± 0.53**	47.09 ± 13.43	0.203 ± 0.009**
Atorvastatin	9	6.28 ± 0.53Δ	1.21 ± 0.41ΔΔ#$	47.52± 12.16	0.132 ± 0.003Δ
HQ	12	6.39 ± 0.51Δ	1.69 ± 0.36Δ	48.74 ± 12.56	0.131 ± 0.005Δ
DS	12	6.86 ± 0.58Δ	1.61 ± 0.42Δ	46.31 ± 14.12	0.148 ± 0.006Δ
HQ + DS	13	5.86 ± 0.41ΔΔ#$	1.15 ± 0.38ΔΔ#$	47.33± 11.27	0.124 ± 0.004ΔΔ#$
Groups	n	APTT (s)	PT (s)	TT (s)	FIB (g/L)
Sham	15	47.3 ± 9.58	14.5 ± 3.41	42.13 ± 9.18	18.04 ± 4.91
MI	11	25.9 ± 6.32**	9.15 ± 2.46**	25.82 ± 7.31**	28.17 ± 5.53**
Atorvastatin	9	29.58 ± 7.53	11.57 ± 4.15	30.18± 6.38	26.27 ± 4.69
HQ	12	29.3 ± 7.28	10.24 ± 3.28	29.76 ± 8.54	25.67 ± 4.92
DS	12	36.4 ± 9.06Δ	12.22 ± 4.15Δ	32.53 ± 7.58Δ	21.71 ± 5.58Δ
HQ + DS	13	38.9 ± 8.27Δ	13.28 ± 3.47Δ	38.23± 5.79Δ	20.47 ± 3.91Δ

Notes: Values are presented as mean ± SD. **p < 0.01 vs sham group; Δp < 0.05, ΔΔp < 0.01 vs MI group; #P < 0.05 vs HQ single treatment group; $P < 0.05 vs DS single treatment group.

Abbreviations: WBV, whole blood viscosity (shear rates: 200 s −1); PV, plasma viscosity (shear rates: 200 s −1); ESR, erythrocyte sedimentation rate; HCT, hematocrit; OTE: oxygen transport efficiency or oxygen delivery index of whole blood = WBV/HCT. APTT, activated partial thromboplastin time; PT, prothrombin time; TT, thrombin time; FIB, fibrinogen.

Discussion

CHD is a major public health concern worldwide. Patients with CHD often experience both physical and mental distress. Repeated MI and long-term hypoxia can induce arrhythmia, cardiac remodelling, myocardial asthenia, heart failure, and eventually endanger life.29 Even during the chronic stable period, lower quality of life is often caused by chronic symptoms including chest tightness, shortness of breath, fatigue, panic, and depression.30 Aspirin and statins are the western medicines prescribed to patients with CHD. However, the side effects are now becoming increasingly obvious. Even a low dose of aspirin can cause indigestion, pain, gastroduodenal ulcer, bleeding, and dizziness while statins can lead to liver injury, myalgia, myositis, rhabdomyolysis, hyperglycaemia, and cognitive impairment.31,32 Therefore, discovering safe and effective medicines to treat CHD is imperative. TCM is famous for its multi-targets, two-way, systematic regulation, and fewer side effects, which are replaceable for those who are ineligible to Western Medicine. HQ and DS are two Chinese herbal medicines, which are frequently used for treating CHD, alone or in combination in the clinic.33 However, the synergistic effects and mechanical actions of HQ and DS on CHD have not been well studied.

In this study, through network analysis, we determined that HQ and DS share the biological processes of nutrient metabolism, cell proliferation, circulatory system homeostasis, inflammatory response, and oxidative stress. The specific biological processes of HQ include metabolism, cell proliferation, fibrosis, inflammatory response, and oxidative stress. The specific biological processes of DS involve lipid and glucose metabolism, circulation system homeostasis, and mood stability. The experimental evidence indicated that HQ and DS exert synergistic effects on improving cardiac function, alleviating myocardium fibrosis, regulating hyperlipidaemia, and hyperviscosity syndrome. HQ exhibits specific advantages on preventing cardiac remodelling after MI, such as reducing left ventricular dilatation and myocardial cell hypertrophy. DS had specific advantage effects on regulating hypercoagulable and maintain hemodynamic homeostasis.

Hyperlipidaemia, along with hypertension, diabetes, and smoking is a major risk factor of CHD.34 Lipid accumulation, inflammatory response, oxidative injury, and fibrosis in the arterial wall is the major pathological basis for CHD.35 Following MI, the modification of the ventricular structure and shape is defined ventricular remodelling (VR). MI induces an intense injury response that ultimately generates a collagen-dominated scar. The excessive fibrotic process is often sustained in a manner detrimental to optimal recovery, resulting in adverse remodelling and impaired cardiac function after MI.36 Excessive collagen proliferation and deposition in both the infarct and non-infarct zone, myocardial apoptosis in the MI zone, myocardial hypertrophy in the non-infarct zone, and the gradual enlargement of the ventricular chamber leads to haemodynamic changes, generating heart failure.37,38 Hypercoagulable status and hyperviscosity syndrome of acute coronary syndromes (ACS) also contributed to hemodynamic changes. The heightened levels of tissue factor and fibrinogen are related with a “hypercoagulable status” and present a higher incidence of ischemic complications after ACS.39 Cytokines, particularly circulating IL-1β, IL-6, and IL-8 are upregulated in systemic and chronic inflammatory conditions. Hypercoagulability is an important hallmark of inflammation, and these cytokines are critically involved in abnormal clot formation, erythrocyte pathology, and platelet hyperactivation, and these three cytokines have known receptors on platelets.40 Drugs of anti-coagulants, anti-hyperviscosity, anti-platelet, and anti-thrombotic should be considered as haemorheological due to the activities in maintaining blood fluidity. The herbal anticoagulant drugs exhibit fewer side effects and better tolerability and, probably, their selected use in patients with a “hypercoagulable status” may improve the clinical outcome after MI.41 Plasma and blood viscosities significantly increase in hyperlipidaemic patients. The hyperviscosity syndrome caused by chylomicronaemia is considered the underlying reason for some of the symptoms.42 Hyperviscosity syndrome is prone to a lower capacity for oxygen delivery to tissues and reduced the efficiency of triglyceride (TG) clearance rate in hyperlipidaemia patients.43 Therefore, regulating hyperlipidaemia, preventing VR, or improving haemodynamics are critical factors for preventing heart failure after MI.

Our findings indicate that HQ combined with DS had synergistic effects on regulating hyperlipidaemia, preventing VR, improving hemodynamic, and thus preventing heart failure after MI. The synergistic effects of HQ and DS may result from their shared biological processes of improving tissue energy metabolism, promoting myocardial cell proliferation, maintaining circulatory system homeostasis, and inhibiting the inflammatory response and oxidative stress. Compared with Atorvastatin, which exerts significant effects on regulating hyperlipidaemia, HQ combined with DS significantly increased the survival rate of MI rats. Data from clinical trials have demonstrated the efficacy of pharmacologic interventions using statins in reducing the risk of CVD events and total mortality in the ever-growing pool of patients as secondary prevention.44 However, statins have been associated with many serious side effects and are unable to lower CVD or total mortality.45,46 In this study, the mortality rate in the Atorvastatin group was higher than in the untreated MI group. We deduce this may be attributed to the side effects of statins, eg, muscle injury, liver injury, and hyperglycaemia, which might be fatal in the acute phase of MI rats because of the left ventricular myocardium under the infarction and hypoxia conditions.

HQ had specific advantage effects on alleviating cardiac remodelling in terms of cardiac hypertrophy and left ventricular enlargement after MI. This may result from its specific function on promoting cardiac metabolism and myocardial cell proliferation, and inhibiting fibrosis, the inflammatory response, and oxidative stress. Metabolic pathways integrate to support tissue homeostasis and to prompt changes in the cell phenotype. The heart consumes relatively large amounts of substrate not only to regenerate ATP for contraction but also to sustain biosynthetic reactions for the replacement of cellular building blocks. The cardiac metabolic inefficiency and loss of coordinated anabolic activity metabolism that occurs during pathological stress (eg, MI, pressure overload) are emerging as proximal causes of pathological VR.47 Oxidative stress due to reactive oxygen species (ROS) and the inflammatory molecules IL-1β, IL-6, TNF-α, and NF-kB, participates in several aspects of cardiac remodelling after infarction that includes cardiomyocyte apoptosis, fibrogenic responses, and hypertrophy.48

DS had the specific advantage in maintaining circulation system homeostasis and mood stability. In this study, DS significantly reduced fibrinogen levels, and prolonged PT, TT, and APTT time with specific beneficial effects on regulating hypercoagulable status after MI. Nowadays antithrombotic therapy in the form of antiplatelet plus anticoagulant drugs, represents the main treatment of ACS, and the prognosis of ACS patients is significantly improved, which is particularly helpful in the acute phase of ACS.39 Most cardiovascular events result from a thrombotic complication of atherosclerotic lesions. In arterial vessels such as the coronary bed, an interrelationship of haemostatic, coagulation, and fibrinolytic factors is implicated. Fibrinogen circulates in plasma as a dimer and plays an important role in coagulation and haemostasis as well as inflammation, and is a well-established risk factor/marker for cardiovascular diseases.49

The limitations of this study can be attributed to three aspects. Firstly, patients with CHD often experience both physical and mental distress. According to the network analysis, DS had the biological process of maintaining mood stability. It would be meaningful if the concentration of serotonin, dopamine, and melatonin in the serum or brain would be studied in the future. Secondly, conventional doses of HQ and DS were used in this study. Herbal medicine often has a positive dose-effect relationship.50 A high dose, conventional dose, and low dose of HQ and DS on treating CHD should be defined in the future. Thirdly, some signalling pathways, such as transforming growth factor β/Smad pathways, which are closely associated with myocardial hypertrophy, remodelling, and fibrosis should be further studied.51,52 This would help to clarify the underlying mechanism of HQ and DS activity on CHD.

Conclusion

HQ combined with DS exerted synergistic effects on regulating hyperlipidaemia, preventing VR, improving haemodynamics, and thus preventing heart failure after MI. HQ had the specific advantage of alleviating cardiac remodelling after MI, while DS specifically alleviated hypercoagulability status after MI. Our results show that HQ and DS not only achieve synergistic effects but also exert complementary effects on MI. We recommend the combination of HQ and DS in treating CHD.