Ameliorative Effects and Mechanism of Buyang Huanwu Decoction on Pulmonary Vascular Remodeling: Network and Experimental Analyses

Hindawi Oxidative Medicine and Cellular Longevity Volume 2021, Article ID 4576071, 13 pages https://doi.org/10.1155/2021/4576071

Research Article Ameliorative Effects and Mechanism of Buyang Huanwu Decoction on Pulmonary Vascular Remodeling: Network and Experimental Analyses

Yucai Chen ,1 Lidan Cui ,1 Can Wang ,2 Jianing Liu ,2 and Jian Guo 1

1School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China 2School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100029, China

Correspondence should be addressed to Yucai Chen; [email protected] and Jian Guo; [email protected]

Received 25 May 2021; Accepted 30 July 2021; Published 13 August 2021

Academic Editor: Albino Carrizzo

Copyright © 2021 Yucai Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Pulmonary hypertension (PH) is a severe and progressive cardiovascular disease. Its pathological mechanism is complex, and the common pathological feature is pulmonary vascular remodeling. The efficacy of existing therapeutic agents is limited. Traditional Chinese medicine (TCM) has its unique advantages in the prevention and treatment of complex diseases. In this study, the approaches of network pharmacology combined with biological verification are employed to explore the role of Buyang huanwu decoction (BYHWD) in the treatment of PH. The active ingredients in BYHWD were first screened based on the ADME properties of the compounds. In turn, the mean of data mining was utilized to analyze the potential targets of BYHWD for the treatment of PH. On this basis, a series of interaction networks were constructed for searching the core targets. The genes including AKT1, MMP9, NOS3/eNOS, and EGFR were found to be possible key targets in BYHWD. The results of enrichment analysis showed that the targets of BYHWD focused on smooth muscle cell proliferation, migration, and apoptosis, which are classic biological processes involved in pulmonary vascular remodeling and are closely related to the PI3K-Akt-eNOS pathway. The methods of biological experiments were adopted to verify the above results. The present study elucidated the mechanism of BYHWD in the treatment of PH and provided new ideas for the clinical use of TCM in the treatment of PH.

1. Introduction Various kinds of complementary and/or alternative medicines have attracted growing interest in the long-term treat- Pulmonary hypertension (PH) is a progressive vascular dis- ment of complex diseases with approved curative therapeutic ease that affects the small pulmonary arteries and is charac- effects. Relevant studies have shown that the traditional Chi- terized by heightened proliferation and apoptosis resistance nese medicine (TCM) and active natural products generate of pulmonary arterial smooth muscle cells (PASMCs) [1]. attractive effects on PH treatment and rehabilitation. Max- Elevated pulmonary vascular resistance results in increased ingxiongting mixture, composed of four herbs, could attenu- pulmonary arterial pressure, right ventricular failure, and ate hypoxia-induced PH and improve right ventricular ultimately death [2]. Current therapeutics for PH in clinic hypertrophy by inhibiting the rho-kinase signaling pathway mainly include endothelin receptor antagonists, prostacyclin [6]. The Qishen Yiqi formula may play a therapeutic role in analogs, and phosphodiesterase-5 (PDE-5) inhibitors [3]. PH through the PI3K-Akt, MAPK, and HIF-1 signaling The aetiology of PH is complex, involving vasoconstriction/- pathways [7]. The Qiliqiangxin formula could inhibit PH- diastolic imbalance, gene mutation, in situ thrombosis, induced right ventricular remodeling by reducing inflammatory response, and oxidative stress [4]. However, mitochondrial-related apoptosis pathway and reversing most drugs highlight the effect on one kind of cell, one cyto- mitochondrial-related metabolic transition [8]. Therefore, kine, or one gene, without considering the complex process the TCM has its unique advantages in the treatment of com- of PH [5]. plex diseases such as PH because of its multicomponent, 2 Oxidative Medicine and Cellular Longevity multitarget, and multipathway mechanism characteristics. It were predicted by importing each Mol2 formatted file into is particularly important to explore the underlying mecha- the Pharmmapper online tool (http://www.lilab-ecust.cn/ nisms of these TCM formulas that have been widely used pharmmapper/) [17]. Based on the normalized fit score, the and are highly effective in the clinic for their promotion top 10 highly matching targets of each were selected. In and optimization. But just using traditional biological exper- addition, the SMILES structural information of compounds imental methods to explore the mechanism of TCM has its was obtained through the PubChem database, and targets limitation. Network pharmacology provides a new way to with a match score greater than 0.5 for each compound solve this problem. By constructing the compounds-targets- were retrieved in the SwissTargetPrediction database based diseases network as well as the subsequent topological on the principle of structural similarity (http://www parameter analysis, the key targets and signaling pathways .swisstargetprediction.ch/) [18]. through which drugs exert their effects can be extracted from “ the complex interaction relationships, providing new ideas 2.2. Collecting the PH-Related Targets. With pulmonary ” for investigating the multitarget drug treatment as well as hypertension as the keyword, the genes associated with PH mechanism [9, 10]. were collected from the DisGeNET database (http://www Buyang huanwu decoction (BYHWD) is a well-known .disgenet.org) and the Comparative Toxicogenomics Data- traditional Chinese medicine formula. The BYHWD is com- base (http://ctdbase.org/) [19]. Duplicate genes in the search posed of seven kinds of Chinese medicine: Huangqi (Radix results were discarded. The UniProt database (http://www Astragali seu Hedysari), Danggui (Radix Angelicae Sinensis), .uniprot.org/) was used to acquire the standard gene symbol “ ” Chishao (Radix Paeoniae Rubra), Chuanxiong (Rhizoma with the organism selected as Homo sapiens. Finally, a total Ligustici Chuanxiong), Taoren (Semen Persicae), Honghua of 901 genes associated with PH was obtained. The intersec- (Flos Carthami), and Dilong (Pheretima), and all of which tion of targets of active components and PH-related targets are recorded in the Chinese Pharmacopoeia. Clinical and was considered to be potential therapeutic targets of preclinical studies indicate the protective functions of BYHWD against PH. The details are described in Table S2. BYHWD on cardiocerebrovascular disease [11–13]. Despite 2.3. Construction of the PPI Network. The STRING database its clinical effectiveness, the mechanism of BYHWD on pul- (https://www.string-db.org/) includes both direct physical monary vascular diseases has not been fully investigated. interactions and indirect functional correlations between In the present study, network pharmacology approaches proteins. It contains not only experimental data, results from were employed to investigate potential pharmacological PubMed abstracts, and other database data but also the mechanisms of BYHWD as a therapy for PH. Targets of com- results predicted by bioinformatics methods [20]. Using this pounds in BYHWD and PH-related genes were acquired from platform, the interaction relationship network among poten- public databases. Then, the protein-protein interaction (PPI) tial targets of BYHWD was constructed. Network visualiza- was obtained to analyze the key targets, and Gene Ontology tion was performed using Cytoscape 3.8.2. Topological (GO) and Kyoto Encyclopedia of Genes and Genomes parameters of these nodes such as the degree, betweenness (KEGG) enrichment was performed to find the potential centrality, closeness centrality, and average shortest path mechanism of BYHWD against PH. In addition, in vivo exper- were analyzed by Network Analyzer in Cytoscape software. iments were conducted to verify the key targets and pathway Among them, the degree value, one of the most predominant predicted in the network. Figure 1 illustrates the workflow. topological parameters in the network, was used to character- ize the degree of nodes. The larger the value of the degree 2. Materials and Methods parameter of a node, the more central the node was considered in the topological network. 2.1. Screening the Components of BYHWD and Predicting Targets of the Components. The components of seven herbs 2.4. Gene-Phenotype Correlation Analysis. The VarElect were collected through the traditional Chinese medicine sys- online platform (https://varelect.genecards.org/), which is tems pharmacology platform (TCMSP, http://old.tcmsp-e an integral part of the GeneCards database, can analyze the .com/tcmsp.php) and TCM Taiwan database (http://tcm correlation of a large number of input genes with a certain .cmu.edu.tw/zh-tw/) [14, 15]. Oral bioavailability (OB), clinical phenotype [21]. The input genes were divided into Caco-2 permeability, and drug like availability (DL) are phenotype-directly related genes and phenotype-indirectly important pharmacokinetic parameters to evaluate the drug- related genes, and the score of correlation was obtained. ability of compounds in the ADME process. The parameters According to VarElect’s calculation method, the correlation OB > of active compounds for selection were set as follows: score in the result is derived from the frequency of a specific 30% DL > 0:18 Caco‐2>−0:4 , , and [16]. Some compounds phenotype in individual GeneCards. The higher the score, were extremely high in content or were reported to have the higher the correlation between the gene and a specific strong pharmacological activities; then, they were included phenotype [21]. In this study, this tool was used to analyze in further evaluation, even if they did not completely meet the correlation between the potential targets of BYHWD the above screening principles. Totally, we collected 93 com- and the phenotype of “pulmonary hypertension.” pounds in BYHWD. The detailed information about chemi- cal constituents of each herb in BYHWD is showed in 2.5. GO and KEGG Enrichment. The Database for Annota- Table S1. The obtained Mol2 type files of ingredients were tion, Visualization, and Integrated Discovery (DAVID, retrieved using the TCMSP database. Next, potential targets https://david.ncifcrf.gov/home.jsp, ver. 6.8) was applied for Oxidative Medicine and Cellular Longevity 3

Buyang huanwu decoction

Compound collection and target prediction

Key targets extraction Pathway enrichment analysis

BYHWD, Saline or Sildenafl

0714 21

Pulmonary hypertension

Histologic examination Monocrotaline Western blotting

Figure 1: A schematic diagram of the integrative strategy combining network pharmacology analysis and biological experiments for investigation of the eﬀect and mechanisms of BYHWD against PH.

GO and KEGG enrichment analysis [22]. The potential ther- 2.6. PH Animal Model and Drug Treatment. The adult male apeutic targets of BYHWD against PH were input into the Sprague-Dawley rats (180–220 g) were supplied by the Vital DAVID platform, and the species was set as “Homo sapiens.” River Laboratory Animal Center (Beijing, China). The ani- The biological processes (BP), molecular functions (MF), and mals were housed under a 12 h light-dark cycle at the condi- ° KEGG pathway enrichment analysis were carried out, tion with a temperature at 21–25 C and the humidity of respectively. Only GO terms and KEGG pathways with P 55%–65%. The rats were allowed access to water and com- values < 0.01 were included. Not only that, the potential ther- mercial food. All the animal procedures were performed in apeutic targets of BYHWD against PH were entered into the accordance with the Guide for the Care and Use of Laboratory KEGG Mapper (https://www.genome.jp/kegg/mapper.html) Animals (National Institutes of Health (NIH), USA). The for further analysis of genes included in key pathways. Based monocrotaline (MCT) was purchased from Sigma-Aldrich, on these results, the top 20 terms with the smallest P value St. Louis, MO. The sildenaﬁl was purchased from Yuanye and their parameters are uploaded to Omicshare (https:// Bio-Technology Co., Ltd (Shanghai, China). The BYHWD www.omicshare.com/) online tools for further visual concentrated granules were obtained from Beijing Tcmages analysis. Pharmaceutical Co., Ltd (Beijing, China). Rats in the MCT- 4 Oxidative Medicine and Cellular Longevity

Astragali Carthami Radix Flos

MOL001645MOL000953 MOL002680MOL001771 MOL000422MOL000098 MOL002694 MOL000006 MOL002695 MOL005448 MOL002698 MOL002687 MOL002706 MOL002442 MOL002707 MOL001831 MOL002710 MOL001443 MOL002712 MOL006812

MOL002717 MOL005384

MOL002719 MOL001956

MOL002721 MOL011782

MOL002735 MOL004492

MOL002736 MOL002208

MOL002757 MOL002202

MOL002773 MOL002157

MOL005421 MOL002151

MOL000033 ESRRG KDM4E MPG CDK6 ADORA1 PLAU FGFR1 PIK3R1 TOP2A ABCG2 APP SYK CCNA2 CD38 MOL002140 Persicae Pheretima ACHE ADORA2A SRC MAPT THRB KDR CXCR1 ADRA2B GSK3B AKR1C1 TOP1 AKR1C3 AVPR2 CA6 Semen MOL000211 MOL002135 ESRRA MAPK8 MAPK10 SLC22A12 ESR2 HSD17B2 GLO1 STS CES1 PDPK1 MAPK14 PLK1 METAP2 PPIA MOL000239 MOL001494 CHEK1 GSTP1 METAP1 CA14 FLT3 F10 MTAP SELP CA3 PARP1 CLC AKR1A1 CFB CAMK2B

MOL000296 MOL000360 MAOB EGFR GRK6 HMGCR IGF1R ALOX5 AXL NEK2 MYLK NEK6 ADRA2A ANXA5 ALOX12 CA5A

MOL000354 CTSB PGR MAPK1 CYP51A1 RIDA AKR1C4 NPR3 PRKACA ADAM17 PRDX5 AR CA1 SAE1U MAOA MOL013129

TYR RTN4R AHR HSP90AA1 CD81 HSD11B1 MMP9 IL2 GSR BCAT2 BCHE LCN2 PKN1 TNKS MOL000371 MOL008457 PDE4B TTR PTPRS PNP PIM1 MMP2 PTK2 NUAK1 AKR1B10 GC AKR1C2 ALB TGFBR1 HCK MOL000378 MOL007022 CYP17A1 EPHB4 DRD4 CYP1B1 NOX4 ADRA2C MET ALK NPC1L1 ABCB1 ARG1 PLG ANG AMY1C

MOL000380 AKT1 APEX1 FCAR PLA2G1B ABCC1 MAPKAPK2 TGFBR2 APOA2 AURKA RORA DDX6 KYAT1 CA4 ADORA3 MOL007018

MOL000387 CA12 PPARD PDE4D NOS3 HSD17B11 PIK3CG DAPK1 SPARC MPO AURKB CDK5R1 ALOX15 KIF11 XDH MOL007012

FAP INSR CDK1 ICAM2 NR1H3 HSD17B1 FUT7 POLB ITGAL CDK5 PAH TNKS2 F2 CMA1 MOL000392 MOL007008 TREM1 CA2 CYP19A1 CA9 CA7 NUDT9 CASP3 FKBP1A BMP2 NR1H2 CANT1 MMP7 CDK2 HSPA8 MOL000398 MOL007005 PPARG GPR35 PYGL MMP8 CTSD MMP12 CA13 PTPN1 SNRPA CASP7 CSNK2A1 MMP3 MMP13 CA5B MOL000401 MOL007004 BACE1 ESR1 GBA AKR1B1

MOL000409 MOL006999

MOL000417 MOL006996

MOL000438 MOL006994

MOL000442 MOL006992

MOL000493 MOL005043 MOL001320 MOL004355 Angelicae Paeoniae MOL001323 MOL002883 Radix MOL001328 MOL002776 Sinensis MOL001339 MOL002714 Radix Rubra MOL001340 MOL001925 MOL001342 MOL001924 MOL001343 MOL001921 MOL001344 MOL001918 MOL001351 MOL000513 MOL001358MOL001371MOL000358MOL000449MOL000492

Chuanxiong Rhizoma

Figure 2: Global herbs-compounds-targets network in the BYHWD. Yellow nodes represent herb components in the formula, 93 red nodes represent compound components in each herb, 200 blue nodes remark the targets of BYHWD from prediction and literature mining, and edges represent interactions between them. induced PH groups received a single subcutaneous injection taining 0.02 mol/L Tris-HCl, 1% Triton X-100, 0.15 mol/L of MCT (50 mg/kg) on day 1. The animals were further NaCl, 1 mmol/L ethylenediamine tetraacetic acid, 1 mmol/L treated with either saline (MCT group), BYHWD solution EGTA, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β- (20 g/kg daily for 21 days, the BYHWD groups), or sildenafil glycerophosphate, and 1 mmol/L sodium orthovanadate). solution (20 mg/kg daily for 21 days, the sildenafil groups, as Western blot assays of PI3K, Akt, and eNOS were carried the positive drug group). The control group was treated with out by standard protocols based on the previous study [24]. saline but did not receive MCT injection. In brief, the supernatants were fractionated by 8% or 10% SDS-PAGE and then electrotransferred onto a PVDF mem- 2.7. Histologic Examination. By the experimental endpoint, brane. The membranes were blocked with 5% BSA in Tris- ° the rats were euthanized. The left lung was removed and buffered saline plus 0.1% Tween-20 (TBST) for 1 h at 37 C. formalin-fixed and paraffin-embedded. Serial sections Then, they were subjected to an immunoblotting assay using (5 μm) were routinely stained using Masson’s trichrome primary antibodies to PI3K (Abcam, ab40755, 1 : 1000), staining. Digital images were obtained at 200x magnifica- phospho-Akt (Abcam, ab81283, 1 : 1000), total-Akt (Abcam, tion by microscopy (Olympus, Tokyo, Japan). The ab179463, 1 : 2000), and eNOS (CST, 32027, 1 : 1000) as medial wall thickness was measured as described previ- well as β-actin (Proteintech, 66009, 1 : 2000) as an internal ously [23]. reference. After incubation with a properly titrated second antibody, the immunoblot on the membrane was visible 2.8. Western Blot. To separate the protein of lungs, the left after development with an enhanced chemiluminescence lung samples were homogenized with whole lysis buffer (con- (ECL) system. Oxidative Medicine and Cellular Longevity 5

MMP2 MMP9 MPO MET NOS3 LCN2 NOX4 IL2 NPR3

IGF1R XDH PARP1 TGFBR2 AKR1B1 HSP90AA1 PIK3CG PPIA ALB GSR PLAU

FGFR1 PIM1 AR PLG

F10 PLK1

MMP3 BMP2 EPHB4 PPARD

EGFR PPARG MAPK8 CDK2

CYP1B1 PTK2

CXCR1 MAPK14 CMA1 SELP

CTSD SPARC

MAPK1 CYP19A1 CDK6 SRC

CASP3 KDR ESR2 TGFBR1

CA9 HMGCR F2 TOP2A GBA ARG1 ABCB1

ANG ABCG2 ALOX5 ACHE ALOX10 AKT AHR ADORA2A

Figure 3: Speciﬁc compounds-targets network of ingredients against PH in the BYHWD. The purple nodes represent the potential therapeutic targets of BYHWD against pH, among which the 20 nodes in dark purple were the core nodes in the network. The red square nodes remark the compounds that are linked to the therapeutic targets, and the darker the red color, the higher its degree value in the network.

2.9. Statistical Analysis. Data are expressed as the mean ± herbs. Part of the compounds, such as ferulic acid SEM of at least three independent experiments and analyzed (MOL000360), chrysanthemaxanthin (MOL004492), and by GraphPad Prism 7 (GraphPad Software, San Diego, CA, stigmasterol (MOL000449), are present simultaneously in USA). Statistical analysis was performed using one-way anal- multiple herbs. Based on structural similarity prediction ysis of variance (ANOVA) followed by Dunnett’s multiple and reverse molecular docking, a total of 200 predicted tar- comparison test. A P value less than 0.05 was regarded as gets of the compounds were collected (Figure 2). The above signiﬁcant. results partly illustrate that there is a common eﬀect for the individual constituent components in the formula. 3. Results 3.2. Interaction Analysis of Anti-PH-Related Ingredients and 3.1. Screening of Active Ingredients and Targets of BYHWD. Targets in BYHWD. Through further analysis, 65 targets According to the properties of compounds such as OB associated with PH among all targets of BYHWD were and DL, 93 components were screened out from seven obtained. Ingredients in BYHWD associated with these 65 6 Oxidative Medicine and Cellular Longevity

AKT1 BMP2 PIK3CG NOS3 ABCB1 TGFBR2 PLK1 XDH

SELP F2 MAPK14 PLAU ARG1 ALOX5 GSR KDR

MPO TGFBR1 PIM1 ANG NPR3 CDK6 AR CXCR1

IGF1R PPIA MAPK1 HSP90AA1 CA9 PPARD ADORA2A EGFR

CASP3 LCN2 CDK2 F10 SPARC AKR1B1 PPARG PARP1

EPHB4 MAPK8 NOX4 MMP2 IL2 ACHE TOP2A CYP1B1

MMP3 CTSD MET AHR MMP9 CMA1 FGFR1 ALB

CYP19A1 HMGCR PTK2 ALOX12 ESR2 SRC PLG GBA

ABCG2

Figure 4: Network biology analysis for PPI constructed with potential targets of BYHWD against PH. In the network, nodes represent targets. Edges (gray) represent PPI interactions. The color intensity of a node is proportional to the value of a degree in the network. targets were acquired. The compounds-targets network was TGFBR1, F2, and KDR (Table 1). Many of these genes also constructed by Cytoscape software. As shown in Figure 3, coincide with the core genes in the PPI network. the C-T network consists of 158 nodes (93 compounds and 65 PH-related targets) and 904 C-T interactions. In order 3.5. Gene Ontology Analysis. The enrichment analysis of to find the main active compounds, the topological parame- potential targets of BYHWD in the treatment of PH was per- ters of the network were further analyzed. The results indi- formed using the DAVID platform to explore the BP and MF cated that the compounds with the highest degree in which these targets were involved. As shown in Figure 6 parameter in the network included quercetin, kaempferol, and Tables S3 and S4, potential therapeutic targets are “ luteolin, 6-hydroxykaempferol, quercetagetin, and baicalein. involved in many biological processes, including negative ”“ These compounds may play a more important role in the regulation of apoptotic process, positive regulation of cell ”“ ”“ anti-PH effects of BYHWD. migration, angiogenesis, positive regulation of cell promotion,”“positive regulation of protein kinase b signaling,”“positive regulation of smooth muscle cell 3.3. Constructing PPI Networks and Analyzing Network promotion,”“response to hypoxia,” and “positive regulation Topological Features. The degree is one of the important of phosphatidylinositol 3-kinase signaling.” The molecular topological parameters, which equals the number of links function terms involved in these targets included “ATP connected to and also the number of neighbors of the node. binding,”“enzyme binding,”“protein tyrosine kinase The value of the degree reflects the importance of the node activity,” and “protein binding.” Obviously, many of the in the network. Using the STRING online tool, the PPI net- biological processes enriched were closely related to the work of BYHWD’s potential targets for the treatment of mechanism of vascular remodeling in PH. PH was constructed. As shown in Figure 4, the targets with higher degree values in the network included AKT1, ALB, 3.6. Pathway Enrichment Analysis. To further clarify the rela- and MMP9. These key targets were more interconnected tionship between targets and the pathways, as described pre- with the other targets and were also involved in multiple sig- viously, the KEGG databases and the KEGG Mapper online naling pathways. So, these core targets may play a more crit- tool were used for pathway enrichment analysis. The top 20 ical role in the anti-PH effects of BYHWD. pathways involving 65 targets were screened. The results showed that the targets were mainly involved in the FoxO signaling pathway, VEGF signaling pathway, PI3K-Akt sig- 3.4. Gene-Phenotype Correlation Analysis of the Targets of naling pathway, TNF signaling pathway, and so on BYHWD. PH is a kind of disease with complex pathogenesis. (Figure 7 and Figure 8). A large number of genes are implicated in the development and progression of PH. These genes vary in the level of signif- 3.7. Experimental Validation. To further investigate the effect icance of the role they are involved in. With the VarElect of BYHWD on pulmonary vascular remodeling, a MCT- online tool, potential targets to the PH phenotype correla- induced PH model was constructed. The Masson trichrome tions were analyzed. Among the 65 analyzed genes, 46 were staining was used to observe the thickness of small pulmo- directly associated with the disease phenotype and 19 were nary vessels. Pulmonary vessels with a diameter less than indirectly associated (Figure 5). Among the genes directly 150 μm were included to calculate vessel wall thickening. associated with PH, the 10 most strongly associated genes The results showed that the thickness of pulmonary small were NOS3, ALOX5, SELP, NPR3, BMP2, ALB, NOX4, vessels was significantly increased after administration of Oxidative Medicine and Cellular Longevity 7

TGFBR1 TGFBR2 MMP9 HSP90AA1 PLG NOS3 PARP1 AKT1 CASP3 NPR3 AR GBA

SRC ACHE GSR PPARD CYP19A1 PIM1 EGFR

CMA1 ALOX12 AKR1B1 KDR

CXCR1

PIK3CG

PPIA ADORA2A HMGCR

MAPK1

XDH

EPHB4 ANG MMP3

FGFR1 ALB F10 MAPK14 CA9

MAPK8

ABCG2 CTSD AHR MMP2

NOX4 PLAU IL2 CDK6 PPARG

MET CDK2 PTK2 PLK1 SELP ESR2 BMP2 F2 ALOX5 ARG1 LCN2 SPARC ABCB1 IGF1R CYP1B1 MPO TOP2A

Figure 5: Correlation between target genes and the PH phenotypes. Green nodes located on the inner circle represent genes that are indirectly associated with PH. The red nodes located on the outer ring represent the directly related genes. The degree of darkness of node color is proportional to its phenotypic relevance.

MCT and was significantly decreased by both BYHWD 4. Discussion treatment and positive drug sildenafil treatment (Figure 9). According to the above mechanistic analysis, the eNOS The prevalence of PH and the incapability of existing vasodi- and PI3K-Akt pathway may be the key targets of BYHWD lation therapy in confronting the complex feature disorder in the treatment of PH. The approach of the biological make the present treatment strategies controversial. TCM experiment was taken to validate the obtained key targets formulas produce synergistic effects by potentially active and pathways. The results of western blot showed that the ingredients acting in a multitarget and multipathway biolog- activation of the PI3K-Akt pathway was significantly ical network, thereby interfering with the occurrence and reduced and the expression level of eNOS was decreased in development of diseases and finally achieving therapeutic MCT-treated rats. After administration with BYHWD, the effects. BYHWD has been widely used in the treatment of levels of the PI3K-Akt-eNOS pathway were significantly many cardiovascular and cerebrovascular diseases. A recent upregulated (Figure 10). study reported that BYHWD could promote neurological 8 Oxidative Medicine and Cellular Longevity

Table 1: Top 10 targets directly associated with PH phenotype. Furthermore, the PPI network and gene-disease phenotype correlation of potential therapeutic targets were analyzed Gene symbol Description Score to find the core targets of BYHWD. AKT1, MMP9, NOS3/e- NOS3 Nitric oxide synthase 3 21.08 NOS, and EGFR may play critical roles in the anti-PH effects ALOX5 Arachidonate 5-lipoxygenase 14.37 of BYHWD. Impaired availability of NO is a key underlying SELP Selectin P 14.25 feature in most forms of PH. NO is produced catalytically NPR3 Natriuretic peptide receptor 3 13.96 from L-arginine by the PI3K-Akt-eNOS pathway and regulates pulmonary vascular function and structure. The decrease BMP2 Bone morphogenetic protein 2 13.68 of eNOS expression and dysfunction will lead to the inhibition ALB Albumin 13.56 of the NO signal in the lung and the disruption of pulmonary NOX4 NADPH oxidase 4 13.42 vasoconstriction/relaxation balance [35]. Dacomitinib, an TGFBR1 Transforming growth factor beta receptor 1 13.32 EGFR inhibitor, can significantly inhibit vascular thickening, F2 Coagulation factor II, thrombin 12.99 collagen deposition, and right ventricular remodeling in PH KDR Kinase insert domain receptor 12.33 rats [36]. Many studies have shown that matrix metallopro- teinases (MMP) play a key role in the disease state, especially in extracellular matrix remodeling and vascular homeostasis recovery and after intracerebral hemorrhage by activating [37]. Increased expression of extracellular matrix proteins is VEGFR2 through the PI3K/Akt signaling pathway [11]. closely associated with MMP activation in pulmonary arteries The active components of BYHWD have multitarget neuro- [38]. There are also close interactions between these targets. protective effects on ischemic stroke, and its mechanism may Vascular remodeling progression involves cell migration be closely related to the improvement of blood supply [25]. A and invasion as well as protein degradation of the ECM in study based on a tag-based digital gene expression profiling which it participates. Upon activation of the PI3K/Akt path- (DGE) system showed that the mechanism of ventricular way, phosphorylated Akt can further activate MMP9, thereby remodeling induced by myocardial infarction by BYHWD playing a role in cell migration and invasion [39]. Dacomiti- treatment may be closely related to transforming growth fac- nib could inhibit cell cycle progression, proliferation, and tor beta receptor-1, junctophilin-2, and monocyte [26]. In migration and enhance cell autophagy in PASMCs induced this study, network pharmacology-based approaches were by hypoxia via the PI3K-Akt-mTOR signaling pathway [36]. conducted to explore the underlying mechanism of BYHWD The approaches of enrichment analysis were further in PH. adopted to analyze the biological processes and signaling Firstly, we predicted the potential targets of BYHWD for pathways involved in potential targets of BYHWD. Multiple the treatment of PH combining potential active ingredients, canonical biological processes closely associated with PH, putative targets of compounds in BYHWD, and targets of such as “negative regulation of apoptotic process,”“positive PH. Through constructing the herbs-compounds-targets regulation of cell migration,”“angiogenesis,”“positive regu- network as well as analyzing topological parameters, the lation of smooth muscle cell proliferation,” and “positive reg- active compounds in BYHWD for the treatment of PH were ulation of phosphatidylinositol 3-kinase signaling,” were extracted, including quercetin, kaempferol, luteolin, 6- discovered in the study. The KEGG enrichment results hydroxykaempferol, quercetagetin, and baicalein. Many showed that the signaling pathways that play therapeutic studies have reported the anti-PH effects of these compounds roles by BYHWD may include “VEGF signaling pathway,” in vitro and in vivo. Studies demonstrate that quercetin can “PI3K-Akt signaling pathway,”“TNF signaling pathway,” inhibit pulmonary arterial endothelial cell transdifferentia- and “FoxO signaling pathway.” VEGF is one of the strongest tion into smooth muscle-like cells by regulating Akt and activators of angiogenesis, which stimulates the migration ERK1/2 pathways and enhance the autophagy of PASMCs and proliferation of vascular endothelial cells, thereby pro- induced by hypoxia through the FoxO1-SENS3-mTOR path- moting angiogenesis. There is a close relationship between way [27, 28]. Quercetin can also interfere with the IRE1α- the overexpression of VEGF and the pathological process of XBP1 pathway and TrkA/kt pathway, leading to reduced PH. Under hypoxic conditions, VEGF is released under the PASMC migration and cell cycle arrest, which in turn ame- regulation of hypoxia-inducible factor (HIF-1), which in turn liorates hypoxia-induced PH [29, 30]. Luteolin can inhibit causes pulmonary vascular inflammation [40]. Akt mediates PDGF-induced proliferation and migration of PASMCs in a cell proliferation induced by various growth factors, which dose-dependent manner via regulating the Hippo-YAP/PI3- have been widely demonstrated. As an upstream regulatory K/Akt signaling pathway [31]. The role of baicalein in the pathway, Akt could be activated by several stimuli, including treatment of PH is the focus of numerous investigators. Bai- VEGF and PDGF. VEGF-induced endothelial migration is calein can delay the progression of pulmonary vascular mediated through the Akt pathway. In terms of vascular remodeling by inhibiting the NF-κB pathway and the cateni- smooth muscle cell apoptosis, FoxOs could induce the n/ET-1/ETR pathway [32, 33]. Baicalein also could attenuate expression of the proapoptotic Bcl-2 family of proteins or MCT-induced PH by inhibiting endothelial-to-mesenchymal upregulated the expression of death receptor ligands such transition [34]. The above results suggest that numerous as Fas ligand and TNFα. Akt can promote the phosphoryla- active ingredients in BYHWD exert effects against pulmo- tion of FoxO, which in turn inhibits the transcriptional func- nary vascular remodeling by regulating multiple signal trans- tion of FoxO, downregulates Bcl-2 levels, and regulates cell duction pathways. apoptosis [41]. It can be seen that BYHWD could exert xdtv eiieadClua Longevity Cellular and Medicine Oxidative ubro nihdtres n h oo ftedt ersne h ereo signi of degree the represented dots the of color the and targets, enriched of number Figure :G nihetaayi a,cnann a ilgclpoesad()mlclrfnto.Tesz ftedt niae the indicated dots the of size The function. molecular (b) and process biological (a) containing map, analysis enrichment GO 6:

GO term Positive regulation of endothelial cellproliferation ligand–activated DNA sequence–specifc binding” GO term “RNA IItranscription polymerase factor activity, Positive regulation of protein Bsignaling kinase Positive regulationof protein phosphorylation Positive regulation of phosphatidylinositol Transmembrane receptr protein tyrosine Negative regulation of apoptotic process Nitric–oxide synthase regulator activity Protein serine/threonine activity kinase Positive regulation of cellproliferation Positive regulation of gene expression Positive regulation of smooth muscle Peptidyl-threonine phosphorylation Positive regulation of cellmigration Peptidyl–tyrosine phosphorylation Serine–type endopeptidase activity Serine–type Positive reugulation of cellgrowth Steroid hormone receptor activity oxygen process metabolic species Peptidyl–serine phosphorylation Protein tyrosine activity kinase Positive regulation of reactive Protein autophosphorylation Protein phosphatase binding Transcription factor binding Identical protein binding Protein phosphorylation Electron carrier activity Protein activity kinase Response to hypoxia MAP kinase activityMAP kinase 3–kinase signaling3–kinase Response toResponse drug Receptor binding cell proliferation Iron ion binding Enzyme binding Wound healing Protein binding Kinase activityKinase activityKinase Heme binding Angiogenesis ATP binding (b) (a) Gene numberGene Gene numberGene .0010.2 0.1 0.00 15.0 12.5 10.0 7.5 5.0 .000 0.10 0.05 0.00 50 40 30 20 10 Rich factor Rich factor ﬁ ac ae nthe on based cance . 0.4 0.3 .50.20 0.15 p value p value 0.00025 0.00050 0.00075 0.00100 0.00125 4.0e–06 8.0e–06 1.2e–05 1.6e–05 P value. 9 10 Oxidative Medicine and Cellular Longevity

Top 20 of KEGG Enrichment p value Transcriptional misregulation in cancer Focal adhesion TNF signaling pathway e Prolactin signaling pathway 4 –05 Chagas disease (American trypanosomiasis) Epithelial cell signaling in Helicobacterpylori infection Colorectal cancer Rap1 signaling pathway 3e–05 PI3K–Akt signaling pathway Hepatitis B Melanoma Pathway Adherens junction 2e–05 Pancreatic cancer VEGF signaling pathway Prostate cancer Progesterone–mediated oocyte maturation 1e–05 FoxO signaling pathway Estrogen signaling pathway Proteoglycans in cancer Pathways in cancer

0.00 0.05 0.10 0.15 Rich factor Gene number 8 12 16 20

Figure 7: The KEGG analysis for the potential targets of BYHWD against PH. The color represents the diﬀerent P values (<0.05), while the size of the circle represents the count.

PI3K-AKT SIGNALING PATHWAY Hypoxia mTOR signaling REDD1 +p pathway 4EBPs eIF4E G�L AMP TSC1 AMPK Rheb mTOR Protein TSC2 +p +p eIF4B synthesis LKB1 +p Raptor S6K1/2 S6 +p +p +p Glucose uptake PKCs Vesicle transport +p PKN Actin reorganization +p Insulin signaling Survival signal, SGK pathway Growth and proliferation +p NO ErbB signaling eNOS pathway VEGF signaling MAPK signaling pathway pathway Cell proliferation Angiogenesis GF RTK Grb2 SOS Ras Raf-1 MEK +p ERK +p DNA repair +p Toll-like receptor +p signaling pathway BRCA1 Pathogen-associated +p molecular patterns TLR2/4 Metabolism (PAMPs) IRS1 GYS B cell receptor PP2A PEPCK Glycolysis/ signaling pathway Rac1 +p Metabolism Gluconeogenesis CTMP GSK3 DNA G6Pace Antigen BCR Syk +p Cell cycle +p Myc PI3K PDK1 +p progression CD19 Class I A DNA Cell cycle JAK/STAT CCND1 BCAP +p progression signaling pathway +p –p p21 PIP3 CDK Cell cycle Cell cycle Cytokine CytokineRJAK AKT p27 +p Cyclin progression –p FoxO signaling Focal adhesion +p pathway p27Kip1 ITGA Class I B HSP90 –p Cell cycle ECM FAK +p FOXO progression ITGB PI3K Cdc37 DNA RBL2 PTEN mTORC2 14-3-3 DNA FasL Cell survival MAGI Bim PHLPP Chemokine +p Bcl-xL BAD Cell survival signaling pathway TCL1 Bcl-2 +p Apoptosis Chemokine, � PI3K Casp9 Cell survival Hormones, GPCR G y +p Bcl-2 Neurotransmitters CREB Cell survival PI (4, 5) P 2 DNA Mcl-1 PTEN PI (3, 4, 5) P 3 +p RXR� NUR77 Bcl-2 Cell survival

+p Bcl-xL NFκB signaling IKK NF �B Cell survival DNA c-Myb pathway +p p53 signaling MDM2 p53 Cell survival pathway 04151 10/3/19 (c) Kanehisa Laboratories

Figure 8: Distribution of the target proteins of BYHWD on the PI3K-Akt pathway. The red nodes are potential target proteins of BYHWD against PH, while the green nodes are relevant targets in the pathway. Oxidative Medicine and Cellular Longevity 11

Control Model 100 ### ⁎⁎ 80 ⁎⁎⁎ 60

(%) 40 20

BYHWD 20 g/kg Sildenafl 20 mg/kg wall thickness Medial 0 MCT – +++ BYHWD (g/kg) 0 0200 Sildenafl (mg/kg) 0 0020

(a) (b)

Figure 9: BYHWD prevents the development of small pulmonary artery hypertrophy in MCT-treated rats. (a) Representative images of the lung sections for intrapulmonary arteries stained with Masson’s trichrome (original magniﬁcation ×200). (b) Medial wall thickness (%) of intrapulmonary arteries was calculated and shown in the bar graph. The results were expressed as the mean ± SEM of three independent experiments (n =3). ###P <0:001 vs. the control group; ∗∗P <0:01 and ∗∗∗P <0:001 vs. the model group.

1.5 1.5 ⁎ ⁎⁎⁎

1.0 1.0 ⁎ PI3K p-Akt # ### t-Akt �-Actin 0.5 0.5 p-Akt / t-Akt p-Akt (fold change) (fold (fold change) (fold PI3K expression 0.0 0.0 Model Control Model

Control MCT – +++ MCT – +++ BYHWD (g/kg) 0 0200 BYHWD (g/kg) 0 0200 Sildenafl (mg/kg) 0 0020 Sildenafl (mg/kg) 0 0020 BYHWD 20 g/kg BYHWD 20 g/kg Sildenafl 20 mg/kg Sildenafl Sildenafl 20 mg/kg Sildenafl (a) (b) 2.0 ⁎⁎ 1.5 eNOS 1.0

�-Actin (fold change) (fold 0.5 eNOS expression eNOS

Model 0.0 Control MCT – +++ BYHWD (g/kg) 0 0200 Sildenafl (mg/kg) 0 0020 BYHWD 20 g/kg Sildenafl 20 mg/kg Sildenafl (c)

Figure 10: Effects of BYHWD on the PI3K/Akt/eNOS pathway. Expression of PI3K and eNOS and phosphorylation of Akt were measured by western blot analysis. Data are expressed as X-fold induction compared to normal control. The results were expressed as the mean ± SEM (n =3-4). #P <0:05 and ###P <0:001 vs. the control group; ∗P <0:05, ∗∗P <0:01, and ∗∗∗P <0:001 vs. the model group. therapeutic effects on PH by acting on multiple targets and of phosphatidylinositol 3-kinase signaling.” And in the multiple signaling pathways. While in the analysis of topo- KEGG enrichment analysis results, the term “PI3K-Akt sig- logical parameters of the PPI network, it was found that naling pathway” also appeared. As mentioned above, there Akt was in the central position in the target network. The is also a tight connection between the PI3K-Akt-eNOS path- results of gene-phenotype correlation analysis suggest that way and other core potential targets, such as VEGF and NOS3/eNOS may be one of the most critical genes in which MMP2. Based on the above experimental results of core tar- BYHWD functions. As can be seen in the GO enrichment get analysis and enrichment analysis, combined with previ- analysis results, the potential targets of BYHWD in the treat- ous studies, we speculate that the PI3K-Akt-eNOS pathway ment of PH were significantly enriched in “positive regula- may be one of the crucial signaling pathways through which tion of protein kinase b signaling” and “positive regulation BYHWD exerts therapeutic effects on PH. 12 Oxidative Medicine and Cellular Longevity

On the basis of network pharmacology analysis, the Supplementary Materials MCT-induced PH model was then constructed to prelimi- narily explore the efficacy and verify the mechanism of Table S1: 93 compounds in BYHWD. Table S2: the potential BYHWD in treating PH. Masson’s trichrome staining results therapeutic targets of BYHWD against PH. Table S3: the showed that the thickening of the pulmonary small vessel was result of top 20 GO (biological process) enrichment analysis. noticeable after rats were stimulated with MCT for 21 days. Table S4: the result of top 20 GO (molecular function) BYHWD treatment can significantly inhibit the vascular enrichment analysis. (Supplementary Materials) remodeling process, which illustrates that BYHWD could ameliorate the PH development from the key pathological References mechanism. This is also consistent with the conclusion from “ network pharmacology analysis that BYHWD may exert [1] S. Fazal, M. Bisserier, and L. 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