key: cord-0068751-dkw3n5ri
authors: Yan, Jiayi; Ruan, Panpan; Ge, Yunxuan; Gao, Jing; Tan, Hongling; Xiao, Chengrong; Gao, Quansheng; Zhang, Zhuo; Gao, Yue
title: Mechanisms and Molecular Targets of Compound Danshen Dropping Pill for Heart Disease Caused by High Altitude Based on Network Pharmacology and Molecular Docking
date: 2021-10-05
journal: ACS Omega
DOI: 10.1021/acsomega.1c03282
sha: 8816e85b0f58de002a5041205c34268f7fdc8478
doc_id: 68751
cord_uid: dkw3n5ri

[Image: see text] Compound Danshen dropping pill (CDDP), a famous Chinese medicine formula, has been widely used to treat high-altitude heart disease in China. However, its molecular mechanisms, potential targets, and bioactive ingredients remain elusive. In this study, network pharmacology, molecular docking, and validation experiments were combined to investigate the effective active ingredients and molecular mechanisms of CDDP in the treatment of high-altitude heart disease. Tan IIA may be the main active component of CDDP in the treatment of high-altitude heart disease via HIF-1/PI3K/Akt pathways.

Chronic mountain sickness is a public health problem in highaltitude areas, such as Qinghai-Tibet plateau located in southwest China, Andean mountains in South America, and other mountainous regions, and more than 140 million people worldwide are exposed to 2500 m above sea level. 1, 2 The heart function of those who usually live at sea level but expose to high altitude for days and months exhibits special pathological characteristics. 3, 4 One of the principal symptoms is highaltitude pulmonary hypertension (HAPH) induced by hypobaric hypoxia (HH), which could subsequently develop into right ventricular hypertrophy (RVH), right heart failure, and ultimately to death in the long term without treatment or drug intervention. 1, 5, 6 However, the mechanism of heart disease caused by HH remained elusive.

Recent research studies show that the oxidative stress process was associated with HH-related heart dysfunction, and the activity of some antioxidants such as superoxide dismutase (SOD) and catalase was decreased under HH conditions, but that of nitric-oxide synthase and malondialdehyde (MDA) was increased. 7 In addition, MAP kinase kinase kinase-2 (MEKK2), an important protein kinase of the MAPK pathway, and hypoxia-inducible factor 1 alpha (HIF-1α) pathway were closely concerned with RVH under HH conditions. 8, 9 However, the detailed molecular mechanism of heart disease caused by HH remains to be determined.

Compound Danshen dropping pill (CDDP) consists of three compositions, namely, Radix Salviae Miltiorrhizae (Danshen), Radix Notoginseng (Sanqi), and Borneolum Syntheticum (Bingpian), and has been widely used for cardiovascular diseases in China, including coronary arteriosclerosis, myocardial ischemia-reperfusion injury, acute myocardial infarction, angina, etc. 10, 11 In addition, it also has been approved by the Australian Therapeutic Goods Administration and is undergoing phase III clinical trial in the US Food and Drug Administration. 12 CDDP can promote adaptation to high-altitude exposure through decreasing the heart rate and myocardial oxygen consumption, 13 but the possible molecular mechanism of CDDP against heart disease caused by HH has been rarely studied.

Network pharmacology is a scientific method that integrates biology, pharmacology, and bioinformatics and comprehensively characterizes intervention of drugs on the disease network. 14 This new bioinformatics method can effectively uncover the molecular mechanism of traditional Chinese medicine. 15 Molecular docking is a powerful technique of docking peptides with the active center of targets and has been widely used in the computer-aided drug design in drug discovery. 16−18 Hence, this approach will help to screen high-affinity energy values between small-molecule compounds in CDDP and the corresponding target receptor. Combining molecular docking and network pharmacology, this study aims to predict the mechanisms and molecular targets of CDDP for heart disease caused by high altitude. The red and violet circle nodes stand for the main active ingredients of Danshen and Sanqi, respectively, and the blue rectangle is the potential target for treating high-altitude heart disease of CDDP.

A total of 520 high-altitude heart disease-related genes were obtained from Genecards and OMIM database (OMIM: 152; Genecards: 368) and further mapped with compound-related genes. Finally, 49 common genes were obtained and imported into Cytoscape software with corresponding compounds to construct the drug−disease-related target network, which contains 94 nodes and 167 edges, as shown in Figure 1 . According to our data, each compound can correspond to one or more targets, and the higher the number of nodes, the greater the importance, indicating that this compound may participate in the treatment of high-altitude heart disease by these corresponding targets. In this network, a total of 48 compounds from DanShen and 7 compounds from SanQi were used, which means DanShen plays an important role in CDDP for the treatment of high-altitude heart disease.

2.3. Target Protein−Protein Interaction (PPI) Network Analysis. The overlapping genes between drug and disease were imported into STRING database to obtain their interaction relationship, and the TSV format file was downloaded to analyzing their interaction based on DC, BC, CC, EC, NC, and LAC parameters using CytoNCA APP. The screening conditions are that the degree, eigenvector, LAC, betweenness, closeness, and network value of genes were larger than the median in all results. After screening three times, six genes were selected as the core target, including PPARγ, VEGFα, IL1β, AKT1, HMOX1, CXCL8, and NOS3. The results are shown in Figure 2 .

2.4. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Enrichment Analysis. GO and KEGG enrichment analyses of 49 common genes were carried out, and top 10 results were screened and displayed in a bubble graph, as shown in Figures 3 and 4 .

The GO results are as follows: (1) The biology process was mainly associated with the vascular process in the circulatory system, regulation of tube diameter, reactive oxygen species metabolic process, apoptosis, and blood pressure (BP). (2) The molecular function was related to steroid hormone and nuclear receptor activity, ligand-activated transcription factor activity, glutathione binding, nitric-oxide synthase, and phosphatase. (3) The cellular component was mainly composed of membrane raft, microdomain, and caveola. The KEGG enrichment result showed that the HIF-1 signaling pathway, PI3K/AKT pathway, p53 signaling pathway, and cytochrome P450 were involved in this process remarkably. 2.5. Target Path Analysis. The pathway map of CDDP for the treatment of high-altitude heart disease was obtained in KEGG Mapper tool, as shown in Figure 5 . Red targets can be regarded as the therapeutic targets of CDDP for the treatment of high-altitude heart disease. The result showed that CDDP treats this disease by the HIF-1 signaling pathway and the vascular endothelial growth factor (VEGF) signaling pathway, with 11 potential effective targets (IFN-γ, RTK, AKT, HIF-1α, VEGF, PAI-1, EDN1, iNOS, eNOS, HMOX1, and Bcl-2). The results are shown in Figure 5 .

2.6. Component−Target Molecular Docking. Autodock tools 1.5.6 was used to validate the binding action of the part C−T−D network by molecular docking. The affinity energy score represents the degree of docking coincidence of molecules; the lower binding energy indicated greater stability. The results suggested that the ingredients of CDDP had good affinity to the protein targets, such as TanIIA, luteolinand, and dehydrotanshinone IIA with BCl-2, VEGF, and SCN5A, respectively. The corresponding binding energies were −8.4, −8.5, and −7.0 kcal/mol, and all of them were less than −5 kcal/mol, which further proved the strong binding ability. The partial docking results are shown in Figure 6 .

2.7. TanIIA Increased the Blood Oxygen Saturation. As illustrated in Figure 7 , during hypobaric hypoxia exposure, SO 2 and PO 2 were decreased significantly compared with the control group (P < 0.0001). However, the treatment with Tan IIA can increase the levels of SO 2 and PO 2 obviously (P < 0.0001).

2.8. Cytotoxicity of TanIIA to Cardiomyocytes. Before investigating the effect of TanIIA in hypoxia conditions to H9c2 and AC16 cells, the cell counting kit 8 (CCK8) assay was applied to detect the cytotoxicity of TanIIA to cardiomyocytes to find a suitable dose. Cell viability of cardiomyocytes was not significant before treatment with different concentrations of TanIIA (0, 0.2, 2, 10, 20, 30, and 60 μM) as shown in Figure 8 . Therefore, 20, 5, and 0.8 μM TanIIA were used in 48 h for the treatment in the following experiments.

2.9. TanIIA Inhibited Hypoxia-Induced Myocardial Enzyme Production. As depicted in Figure 9 , aminotransferase (AST), creatine kinase (CK), creatine kinase-MB (CK-MB), and lactate dehydrogenase (LDH) levels of rat and cell culture media in the model groups showed a more obvious increase than the control group (all P < 0.05), whereas enzyme production was significantly downregulated after the treatment with Tan IIA.

2.10. TanIIA Downregulated Hypoxia-Responsive HIF and PI3K/AKT Pathways. We further explored the molecular mechanism of TanIIA involved in HH-related heart dysfunction in H9c2 and AC16 cells. The HIF and PI3K/ AKT pathways, which were key pathways associated with the effect of TanIIA against HH-related heart dysfunction in our KEGG enrichment results, were analyzed through western blotting. The results show that TanIIA significantly inhibited the expression of HIF-1 and increased PI3K and P-AKT expression. The result is shown in Figure 10 .

Chronic mountain sickness, especially HH-related heart dysfunction remains a public health problem. To date, it has been still a challenge for researchers to discover exclusive drugs for high-altitude heart disease in modern medicine. Increasing experiments and clinical cases suggested that CDDP could be applied to prevent and treat high-altitude heart disease in China 13 but the drug compositions of CDDP are complex, and the specific mechanism of its treatment of high-altitude heart disease still needs to be fully elucidated.

In this study, network pharmacology, molecular docking, and in vitro experimental validation were integrated to reveal bioactive compounds and the molecular mechanism of CDDP for treating high-altitude heart disease. A total of 51 bioactive compounds and 49 common protein targets were selected, and the PPI network showed that PPARγ, VEGFα, IL1β, AKT1, HMOX1, CXCL8, and NOS3 may play an important role in the effect of CDDP against high-altitude heart disease. The top 10 KEGG pathways are shown in Figure 4 , including the HIF-1 signaling and PI3K/AKT pathways. HIF-1, one of the main regulators in hypoxic response, regulates many biological functions, such as immune function, oxidative stress, and metabolic reprogramming. 23 Recently, studies have shown that HIF-1α is a potential therapeutic target in tissue remodeling during physiological adaptation to high-altitude hypoxia conditions. 24, 25 However, the role of HIF-1 in high-altitude heart disease remains to be determined. The PI3K/AKT signaling pathway, which exhibited a higher gene count enrichment among CDDP against high-altitude heart disease's pathways, plays a critical role in the adaptation of tissue cells to environmental hypoxia. 26, 27 Accumulating evidence indicates that Akt was able to activate transportation of HIF-1α to the nucleus and trigger the signaling cascade for survival and PI3K/Akt and HIF-1α pathways, which play a critical role in hypoxia-related diseases, such as rheumatoid arthritis, liver transplant, etc. 28, 29 Flavonoids are polyphenols and are known to have a lot of health promoting effects. For example, Quercetin can decrease blood pressure (BP) and exhibits favorable effects in hypertension. 30 In addition, numerous studies reported that serum lipid levels of moderate hypercholesterolemia patients were decreased after intervention of bergamiol extract and fruit juices, which contain high quantities of flavonoids. 31,32 Tan IIA, a natural flavonoid isolated from Silvia miltiorrhiza is one of the potential targets of active compounds in our screened result. 33, 34 However, to our best knowledge, this is the first study to show that the Tan IIA-HIF-1/PI3K/Akt regulatory network is a novel strategy for the treatment of high-altitude heart disease. The therapeutic effect of CDDP is further suggested by Tan IIA in rats and two types of myocardial cells in the hypoxia model. Blood oxygen saturation (SaO 2 ) and oxygen partial pressure (pO 2 ) were not strongly altitudeindependent, and the levels of SaO 2 and pO 2 were decreased along with the reduced ambient oxygen pressure, leading to subsequent oxygen deprivation of tissues. 35 Furthermore, the levels of AST, LDH, CK, and CK-MB in serum and cell culture media, which are cardiac marker enzymes, were elevated due to cardiac membrane damages in some diseases. 36 In our study, we observed that Tan IIA can increase SaO 2 and pO 2 levels in rat and there were elevated activities of AST, LDH, CK, and CK-MB in serum and cell culture media under hypoxia conditions, but Tan IIA can downregulate the level of these cardiac marker enzymes. Moreover, western blot analysis also showed that Tan IIA significantly inhibited the expression of HIF-1 and increased PI3K and p-AKT expression in vivo and in vitro. Taken together, our study suggested that Tan IIA can rescue heart and myocardial cell injury under hypoxia conditions via HIF-1/PI3K/Akt pathways, which may be a potential molecular mechanism of CDDP in the treatment of high-altitude heart disease.

In this study, network pharmacology, molecular docking, and experiments were combined to investigate the effective active ingredients and molecular mechanisms of CDDP in the treatment of high-altitude heart disease. Further, our findings show that Tan IIA may be the main active component of CDDP in the treatment of high-altitude heart disease via the HIF-1/PI3K/Akt pathway.

5.1. Chemicals and Reagents. H9c2 rat cardiomyoblast cell line was purchased from ATCC (Manassas, VA). AC16, a human cell line cardiomyoblast, was purchased from , and aminotransferase (AST) estimation were obtained from Roche Diagnostics (Germany). Radioimmunoprecipitation assay (RIPA) lysis buffer and the BCA protein detection kit were purchased from cwbiosciences (Beijing, China). Antibodies against PI3K, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and anti-rabbit horseradish peroxidase (HRP)conjugated IgG were purchased from cell signal technique (Boston, MA). Antibodies against HIF-1α, AKT, and p-AKT were obtained from Zen bioscience (Chengdu, China).

Screening. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) were used to screen the active compounds in CDDP, including DANSHEN, SANQI, and BINGPIAN. 19,20 TCMSP, with 499 Chinese herbs recorded, is a unique system pharmacology platform of Chinese herbal medicines, which provides the relationships between drugs, targets, and diseases. 21 The most significant pharmacokinetic parameters in absorption, distribution, metabolism, and excretion (ADME) system including oral bioavailability (OB) and drug likeness (DL) were selected to screen the bioactive compounds in CDDP. Only compounds with the oral bioavailability ≥30% and drug likeness ≥0.18 were selected for subsequent research. Then, the potential targets of active compounds were collected from TCMSP database (http://lsp. nwsuaf.edu.cn/tcmsp.php) and the gene symbols of screened genes were verified by UniProt (http://www.uniprot.org).

5.3. Target Acquisition for Drug and Disease-Related Effects. The target genes associated with high-altitude heart disease were extracted from Genecards (https://www. genecards.org/) and OMIM (https://www.omim.org/). All screened targets were merged and duplicates were removed. The R package R × 64 3.6.1 venny was used to map the chemicals−target−disease (C−T−D) network and to draw Venn diagrams. The common genes of high-altitude heart disease and drug targets were collected, which might be the potential therapeutic targets of CDDP to high-altitude heart disease.

5.4. Construction of Drug Active Ingredients and Disease Target Network. Targets of active compounds and diseases were imported into Cytoscape software (version 3.8.0), and the C−T−D network was constructed. The Network Analyzer Apps software of Cytoscape is used to analyze the topology properties of the network. We also used the degree value of molecules to determine the number of direct neighbors of a node. The large value means that the node plays a highly important role in the network. 22 5.5. Protein−Protein Interaction (PPI) Network Construction and Core Gene Screening. The overlapping genes of CDDP and high-altitude heart disease were put into STRING (https://stringdb.org/cgi/network. version 11.0) with the species limited to "Homo sapiens" and a confidence score of >0.4. The TSV format file was downloaded and imported into Cytoscape. To further screen out the key targets, a series of parameters of Cytoscape plugin CytoNCA, including degree centrality (DC), betweenness centrality (BC), closeness centrality (CC), eigenvector centrality (EC), local average connectivity-based method (LAC), and network centrality (NC). Based on this topological property analysis, the core genes were selected after screening three times. 5.6. GO and KEGG Enrichment Analysis. R software projects were used for gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. T package was applied to convert ID of core genes, and then GO and KEGG were enriched by Cluster Profiler. In this process, statistical significance threshold of enrichment analysis was set at a p value cutoff = 0.05 and a q value cutoff = 0.05 to obtain the key information. Only top 10 items were displayed in the bar graphs. 5.7. Component−Target Molecular Docking. Twodimensional (2D) structures of drug-like molecules were downloaded from PubChem database (https://pubchem.ncbi. nlm.nih.gov/) and then transformed into a PDB format through Chem3D, and saved in PDBQT format as docking ligands in AutoDock Tools 1.5.6 software. The threedimensional (3D) structure of common target proteins was downloaded from the PDB database (https://www.rcsb.org/) and put into AutoDock Tools 1.5.6 software. The water molecules of cocrystallized ligand and structural were removed from the crystal structure and the polar hydrogen atoms were added. The active binding pocket was set up. The grid spacing was 1.000 Å, and other parameters remained default values. Finally, all of the compounds were docked into the prepared protein, and 20 conformations were generated. All docking results were visualized in Pymol 2.3 software.

5.8. Validation Experiments. 5.8.1. Animal Model. All animal experiments were approved by the the intramural Committee on Ethics Conduct of Animal Studies of Academy of Military Medical Sciences, China. Eighteen male and eighteen female Sprague−Dawley rats weighing 200−220 g were housed at microisolator cages and allowed free access to water under standard laboratory conditions. Rats were randomly divided into three groups: control and model group; rats were given 0.9% NaCl by oral administration; TanIIA group received TanIIA by oral administration (10 mg/ kg). After 7 days treatment, rats were subjected to a decompression chamber for 10 days at an equivalent altitude of 6000 m and then specimen and blood were collected. 5.8.2. Blood Gas Withdrawal and Analysis. After hypoxia, the abdominal cavity of the experimental animals was quickly opened and the arterial blood was extracted with a syringe. The samples were immediately analyzed with an ABL90 (Radiometer) blood gas analyzer (an automated, compact analyzer used in clinical and laboratory settings that requires a minimum sample size of 65 μL). SO 2 and pO 2 were directly measured.

5.8.3. Cell Culture. H9c2 and AC16 cells were cultured in DMEM and DMEM/F12 (1:1) basic(1X), respectively, and supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (Lonza, Levallois, France). Cells were randomly divided into five groups: control group and model group, cells cultured in media; three TanIIA group, cells treated with 0.8, 5, and 20 μM TanIIA in media, and then control group cells were cultivated under normoxia conditions, model group and three TanIIA group cells were cultivated at 1% O 2 /94% N 2 /5% CO 2 hypoxia environment for 48 h. 5.8.4. CCK8 Assay. H9c2 and AC16 cells were plated into 96-well plates at 8 × 10 3 and 1 × 10 3 cells/well, respectively, and treated with different concentrations of TanIIA (0, 0.2, 2, 10, 20, 30, and 60 μM) for 48 h. The CCK8 reagent was added to the wells and incubated at 37°C for 2 h and then the absorbance was measured at 450 nm wavelength. 5.8.5. Measurement of Myocardial Enzyme. The serum and cell culture media levels of creatine kinase (CK), creatine kinase-MB (CK-MB) isoenzymes, aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were measured by the automatic biochemical analysis apparatus (Roche Diagnostics, Germany). 5.8.6. Western Blotting Assay. Total protein was extracted with RIPA lysis buffer and the BCA protein detection kit was used to measure protein concentrations. Thirty micrograms of the protein lysate was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) transferred onto poly(vinylidene fluoride) membranes. After blocking in 5% bovine serum albumin for 3 h at room temperature, the membranes were incubated with primary antibodies including AKT, p-AKT, PI3K, HIF-1α, and GAPDH at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)linked secondary antibody at room temperature for 1 h. The omin-enhanced chemiluminescence detection kit was used to detect and visualize protein bands.

5.9. Statistical Analysis. Data are expressed as mean ± SD. One-way analysis of variance (ANOVO) test was used to evaluate the difference between groups, and p < 0.05 was chosen as the threshold for statistical significance. Statistical analysis was performed using GraphPad Prism version 8 (GraphPad Software, La Jolla, CA).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03282.

Main active components and corresponding targets of CDDP (PDF)

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